Characterization of grapevine endophytic microbes and field evaluation of bioformulations against powdery mildew disease with residue-compliant grape production  

Characterization of grapevine endophytic microbes and field evaluation of bioformulations against powdery mildew disease with residue-compliant grape production | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Characterization of grapevine endophytic microbes and field evaluation of bioformulations against powdery mildew disease with residue-compliant grape production Somnath K. Holkar, Shweta C. Nanekar, Sagar B. Bhondave, Sakshi J. Patil, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6602877/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Grapevine ( Vitis vinifera L.) plays a major role in producing table grapes, raisins, juice, and wine in the global market. In the present study, 13 endophytes were isolated, purified, and identified by sequencing the ITS and 16S rRNA regions. Based on ITS sequence information, the fungal endophytes viz. , CSBY-2, CSBY-8, and MCBY-2, were identified as Trichoderma asperellum , whereas CSBY-4, MCBY-1, and DRRS-1 were identified as T. asperelloides , Cytospora sp., and T. viride , respectively. Similarly, the bacterial endophytes viz ., Sauvignon Blanc-2, -5, CS2, and RF1, were characterized as Bacillus subtilis . The RT7 and RB-1 were recognized as B. mojavensis and B. licheniformis , respectively, by sequencing 16S rRNA. The fungal endophytes were formulated into powder using flattened rice as a carrier, and bacterial isolates were used as liquid formulations. Bioformulations of these isolates were evaluated against grapevine powdery mildew disease during the 2022-23 and 2023-24 crop seasons at the experimental fields at ICAR-NRCG, Pune. In field evaluation of endophytic Trichoderma , Bacillus and Cytospora species formulations during the 2022-23 and 2023-24 crop seasons, the highest percent disease control (PDC) on leaves was recorded in treatment, Bacillus subtilis (RF-1: 63.41%) followed by B. subtilis (SB2: 61.57% and 58.11%), Bacillus sp. (GB2: 61.04%), B. subtilis (SB5: 59.66% and 56.65%), and Trichoderma asperellum (MCBY2: 51.07% and 60.93%). The highest PDC on bunches was observed in SB2 (64.56% and 44.97%), SB5: (56.18% and 46.07%), and MCBY-2: 55.88% and 67.7%) isolates. Moreover, all the fungal and bacterial formulations reduced the fungicide residues in the grape berries. The study provides important insights into managing grapevine diseases sustainably and suggests integrating endophytic microbes into traditional farming practices to enhance grape quality and productivity. Biocontrol grapes powdery mildew endophytes Trichoderma asperellum Bacillus subtilis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Grapevine ( Vitis vinifera L.) is a significant fruit crop grown worldwide. Grapevine is a cultivation aimed at various purposes like fresh consumption, raisins, juice, and wine production. Consumption of grapes benefits human health due to their nutritional and medicinal properties (Garcia Conesa et al. 2023). Worldwide, grapes are grown on around 7.5 M hectares by 79 million MT of final produce (FAO, 2023; APEDA, 2023 -24). Grapes are planted on about 171 thousand ha with a total output of 3781 thousand MT in India (FAO, 2023; APEDA, 2023 -24). In India, more than 90% area is devoted to table grapes, followed by raisin and wine production. (Sawant et al. 2017 ; Kanitkar et al. 2020 ). During 2023–2024, India exported about 2,63,075.67 metric tons of grapes costing around 305.66 million USD to many countries (APEDA, 2023 -24). Grapevine cultivation worldwide is becoming significant because of the incidence of several biotic constraints under changing climatic conditions, causing huge losses for grapevine industries by reducing quality and financial loss (Bove et al. 2022 ). Among the biotic constraints, powdery mildew caused by Erysiphe necator is an economically important disease affecting grapevine production and productivity for domestic and international markets (Gadoury et al. 2012 ; Vinothini et al. 2014 ; Xu et al. 2023 ). The commercially cultivated grapevine belongs to the genus Vitis and the Vinifera species is widely known for its susceptibility against powdery mildew disease (Fedorina et al. 2022 ). Throughout the growing season, powdery mildew affects the leaves, canes, tendrils, inflorescences, rachis, and bunches during dry and cool conditions (Gadoury et al. 2012 ). Initially, yellow spots appear on the young leaves, and later white-greyish powdery masses appear on both sides of the older foliage. Diseased young foliage shows curling and inflorescence results in poor fruit set, leading to premature fruit drop. The severely affected berries show cracking and rot (Thind et al. 2004 ; Gadoury et al. 2011 ). The E. necator survives in humid conditions, with temperatures ranging from 20-30 o C, relative humidity of more than 45%, and intermittent rainfall (Gadoury et al. 2012a ). Management of powdery mildew becomes very essential to avoid crop loss. Therefore, the application of chemicals becomes important during favourable climatic conditions. Constant use of fungicides results in the advancement of resistance in phytopathogens, deposits on the bunches, and their harmful effect on living creatures (Pathak et al. 2022 ). To address these issues, the application of potential microbes that originated as epiphytes, endophytes, and rhizospheric microbes having multiple beneficial effects, especially disease management, and bioremediation of residues, needs to be explored (Holkar et al. 2023 , 2024 ). This approach was found to be environment-friendly and sustainable for grapevine disease management. Grapevine powdery mildew disease has been managed efficiently by beneficial biological control agents (BCAs) viz ., Ampelomyces quisqualis , Trichoderma asperelloids T. harzianum, T. afroharzianum, Bacillus subtilis , and Brevibacillus brevis (Sawant et al. 2011 ; Sawant et al. 2017 ; Sawant et al. 2020 ; Caffi et al. 2022 ; Avan et al. 2023 ; Jena et al. 2023 ; Saha et al. 2023a ). Endophytic microbes are more diverse bio-resources that benefit host plants as biocontrol agents, provide tolerance to various stresses under changing climatic conditions, and promote plant growth (Jia et al. 2016 ; Wu et al. 2018 ; Xie et al. 2018 ; Joshi et al. 2019a , b ; Holkar et al. 2023 , 2024 ; Saha et al. 2023 ; Kaya et al. 2024 , 2025 ). Grapevine endophytic microbes have been widely used in managing other grapevine diseases which was recently reviewed (Holkar et al. 2024 ). Little or no data is presented on applying fungal endophytes for powdery mildew disease management and producing residue-compliant grapes worldwide. Therefore, the present investigation was undertaken to evaluate a diverse group of fungal and bacterial endophytes against powdery mildew management and residue bioremediation. This study will help to create awareness among grape growers to increase the share of BCAs, especially the multiple effects of endophytic microbes for quality and low-input cost production. MATERIALS AND METHODS Sample Collection The experimental field of ICAR-NRC for Grapes, Pune (8° 29′ 570′′ N latitude and 73° 59′ 168′′ E longitude) is at 559 meters altitude from sea level. Different plant parts were collected from grapevine cvs. Manik Chaman (Berries), Dogridge rootstock (stem), Sauvignon Blanc (leaf), Cabernet Sauvignon (leaf, roots, and berries), and wild relative Vitis rotundifolia (leaf). The healthy berry samples (mature and immature) of the grapevine cv. Muscat were collected from the Grape Research Station (GRS), experimental field, Theni, Tamil Nadu state (coordinates: 90 45’ 26.685’’N latitude and 770 20’ 58.737’’E longitude) at 435.05 meters. Isolation and Purification of Endophytes The collected various plant parts were cleaned using fluent tap water, soaked in a 3% NaoCl (sodium hypochlorite) for three min, and later washed using 70% ethyl alcohol for 30 sec. After rinsing with sterile distilled water (SDW) four times and blotting dry on sterile filter paper (Whatman, Himedia, Germany) for five min. Cleaned leaves and stems were segmented into five mm-sized pieces using a sterilized cork-borer and scissors, respectively. Sterilized samples (leaf and stem) and berries, four samples of each, were inoculated in each Petri dish poured with PDA and NA medium (Himedia, India), approximately 20 ml in each plate, along with 0.2 g/l chloramphenicol. These Petri dishes were then kept for incubation for four days at 26 °C + 2 o C for growth. After the incubation period, endophytes obtained from the plates were identified based on their specific growth characteristics on the media (Joshi et al., 2019). The fungal endophytes were purified using the hyphal tip of endophytic fungi by subsequent sub-culturing five times on new Petri dishes poured with PDA medium. Similarly, the ooze out bacteria from leaf cuts were purified on new Petri dishes containing NA medium (Joshi et al. 2019a). Spore morphologies and colony characteristics of Trichoderma isolates were studied based on phialide cell arrangement. The mother cultures of all isolates were preserved in sterile broth containing 50% glycerol and stored at -80°C for further use. Microscopic Detection of Fungal Endophytes Isolated endophytes were prepared for microscopic examination using a sterile glass slide, and needle, and stained with methyl blue and lactophenol, and observed under a microscope (Leica model: DM750, Microsystems, Germany) with 10X, 40X, and 100X magnifications. For optimal clarity at 100X magnification, the immersion oil technique was used. A microscopic study was conducted as mentioned by Jaklitsch et al. (2012). The purified EB strains were subjected to morphological characterization (Zhao et al. 2016; Urbez-Torres et al. 2020). Morphological and Biochemical Characterization of Bacterial Endophytes Four EB isolates used in this study were earlier characterized for their morphological, biochemical, and molecular characterization (Holkar et al. 2024). In contrast, the other three EB strains were characterized in the present study for colony characteristics, Gram’s reaction (Hucker and Conn 1923), biochemical parameters, and 16S rRNA sequence data. All characteristics of these isolates were studied by referring to Bergey et al. (1939). Each test was carried out twice, with three replications. DNA Isolation and PCR Amplification of Fungal and Bacterial Endophytes Extracted the total genetic material from six fungal endophytes from 100 mg of the 7-day-old culture of endophytes using the protocol described by the DNeasy Plant Mini Kit (Qiagen, Germany). Further, isolated endophyte mat (fungi) was ground to a fine dust using a sterilized mortar-pestle with liquid N 2 . One milliliter of aqueous phase was added to another 1.5 ml microcentrifuge tube and centrifuged at 14,000 rpm for 5 min. The aqueous phase was transferred to a sterilized 1.5 ml microcentrifuge tube, and the subsequent steps were described. DNA elution, the total genetic material was stored in a deep freezer (-20°C). The nuclear ribosomal DNA ITS (internal transcribed spacer) endophytes were amplified using the universal primer pair ITS1 and ITS4 (White et al. 1990). PCR was carried out using a PCR machine (GeneAmp 9700). Total 25 µl reaction recipe composed of 0.3 µl of each primer (100 ng), 0.75 µl of 10 mM dNTPs, 0.25 µl of Taq DNA polymerase (Thermo Fisher Scientific, USA; 5 U/µl), 4.5 µl of DNA template (~2.5 µg), and 2.5 µl of 10× buffer. The PCR profile includes 10 min denaturation at 94°C at the initial stage, later 35 rounds of subsequent denaturation for 30 seconds at 94°C, 55 seconds at 55°C annealing, and 1 min at 72°C for extension. A 10-minute incubation at 72°C for final extension. Analyzed the amplicons using 1× TAE buffer (90 mM Tris-acetate, 2 mM EDTA, pH 8.0), 1% agarose gel electrophoresis stained with 0.01% ethidium bromide (0.5 µg/ml). Cleaned the amplicons using PCR Purification Kit (QIAquick, Qiagen, Germany) according to the protocol. Amplicons from six endophytes were sent for the double-pass Sanger method at GenOmBio Technologies Pvt. Ltd. Pune, Maharashtra, India. Total DNA was isolated from of 24 h old grown seven endophytic bacterial strains with the help of the producer's protocol (Himedia, India). Eluted total DNA was stored in a deep freezer (-20°C). The total DNA was amplified by PCR using the 27F and 1492R universal primer pair corresponding to the 16S rDNA gene (Lane, 1991). The same PCR machine was used for bacterial endophytes with the same PCR reaction recipe, except DNA template. The PCR profile for amplification of 16S rDNA was like ITS primers except annealing temperature at 57 °C for 30 s, and an extension at 72 °C for 90 s. Amplicons were cleaned up and submitted for double-pass Sanger sequencing. Phylogeny Among the 13 isolates, 12 isolates showed desired amplification and were subjected to sequencing from GeneOmbio, Technologies Pvt. Ltd., Pune, Maharashtra State, India. Nucleotide sequences were assembled in the SeqMan program (Swindell and Plasterer, 1997). The analyzed sequences were submitted to the NCBI GenBank. The BLASTn analyses (https://blast.ncbi.nlm.nih.gov/) were carried out for sequence homology of six EB strains and six FEs. The sequence identity matrix and alignment were carried out using the ClustalW program using BioEdit software version 1.7 (www.bioedit.software.informer.com). The evolutionary history was inferred using MEGA11 software (Kumar et al., 2016; Tamura et al., 2021) by the Neighbour-Joining method (Saitou and Nei, 1987). A 1000 bootstrap test was undertaken to determine replication percentage (Felsenstein, 1985). The sequences from NCBI GenBank were used for comparative analyses as presented in the phylogenetic tree. Development of Bioformulations Based on the in vitro antagonistic effect of these endophytic microbes on the mycelium growth of Colletotrichum gloeosporioides , a causal pathogen of anthracnose in grapevine. Therefore, formulations of six FEs and seven BEs were developed and evaluated for the Erysiphe necator under field conditions, considering direct antagonism of the target pathogen. For the development of the bioformulation of FEs, flattened rice was used as a carrier and applied under field conditions to test their efficacy against powdery mildew disease on grapevine cv. Sarita Seedless during the 2022-23 and 2023-24 crop growing seasons. Briefly, the procedure of preparation of Trichoderma formulation is as follows. The MnSO₄ (0.4 g), D-(+)-Glucose anhydrous (20 gm), CaCl₂ (0.4 gm), and MgSO₄ (0.4 gm) were dissolved in 1 L of distilled water. Flattened rice 1 Kg was soaked in double-distilled water, and the chemical solution prepared using the chemicals described above was mixed thoroughly. The mixture was sterilized at 121°C under 15 psi for a duration of 40 min in a vertical autoclave machine. After sterilization, the flattened rice was allowed to cool and transferred to sterile aluminum trays of approximately 30 cm x 20 cm size to ensure uniform drying and inoculation. A week-old pure cultures of Trichoderma species were inoculated onto the flattened rice, and the trays were kept for incubation at 28–30°C with an absolute moisture of 80–85% for two weeks. Sufficient moisture, 60% - 70%, was maintained in the inoculated trays by spraying double-distilled water. After two weeks of incubation, Trichoderma colonized the flattened rice, and mycelial and sporulation growth were observed. These trays were kept for sun drying for 2-3 h. Colonized dried substrate ground to fine dust particles, with the help of a homogenizer, and stained homogeneous fine powder having 1×10 9 spores/g. It was used for spraying under field conditions for powdery mildew management. The reproducibility of each formulation was recorded. Similarly, for the bacterial formulations, single bacterial colonies were inoculated in a nutrient broth medium for mass production from the bacterial colonies grown on an NA medium. This was incubated at 28 + 2 o C for 48 h. The liquid formulations with 0.5 OD were used for the field sprays. The spore count of the liquid formulation was maintained at 1 × 10 11 CFU/ml. Field Evaluation of Fungal and Bacterial Bioformulations The present investigation was conducted at the institute's experimental farm during the 2022-23 and 2023-24 crop seasons to evaluate the efficacy of isolated endophytes for powdery mildew disease in grape cv. Sarita seedless. Bioformulation evaluation was based on the natural occurrence of the disease. The climatic conditions during both seasons were conducive to the natural occurrence of the disease. The experimental cultivar was eight years old with black soil (Vertisol) having pH of 7.5-7.6. The sprays were undertaken when the first symptoms of the disease were observed on vines. The sprays were scheduled weekly; the vines were treated with sulphur 80% WDG @ 2 g/L as a positive control. For each treatment, three vines were used with three replications by applying a Randomized Block Design (RBD). A knapsack sprayer was used for uniform coverage of endophytic bioformulations on leaves and berries. The percent disease index was observed before scheduling the first spray till one week after the last spray by applying a 0–4 rating score (Horsfall and Heuberger 1942). A total of 100 random leaves per replication (10 canes and 10 leaves per cane) were observed for the disease and assigned a 0-4 score based on the symptoms and severity. The percent disease index (PDI) was calculated using the score data (Wheeler, 1969). The disease ratings had also been recorded on the bunches using a similar scale used for foliar symptoms. A total of 20 bunches were observed per replication. Sum of all numerical ratings Percent Disease Index = --------------------------------------------------------------------- x 100 Number of leaves/bunches observed x maximum grade The percent disease control (PDC) is calculated as under: PDI in untreated control – PDI in treatment PDC = ------------------------------------------- x 100 PDI in untreated control Physical and Biochemical Characterization The harvested grape bunches of the grapevine cv. Sarita Seedless was subjected to the estimation of physical and biochemical characteristics carried out during the 2022-23 and 2023-24 crop seasons. The physical characteristics, like 50-berry weight, bunch weight, skin thickness, rachis length, number of berries per bunch, berry length, berry diameter, total soluble solids (TSS), acidity, pH, pedicel length, physiological weight loss (PWL), and yield were estimated (Satisha and Somkuwar 2019; Thosar et al. 2020). The biochemical analysis included the evaluation of phenol, tannin, reducing sugars, protein, proline, carbohydrates, and antioxidants (Somkuwar et al. 2024). Physiological Weight Loss (PWL) Shelf-life analysis of a grapevine sample harvested from cv. Sarita seedless was calculated based on a weight-loss basis. Weight loss was calculated by recording the initial and final weight by applying a formula: Weight loss = [(RI-RII) ÷ RI] x 100 where, RI= Initial Weight and RII– Final Weight Chemical Reagents and Preparation of Stock Solution for Pesticide Residue Extraction The certified reference material (CRM) of selected pesticides (98.20-99.50% purity) was used (Augsburg, Germany). Analytical quality ethyl acetate (99.8%), acetonitrile (98.4%), LC-MS grade methanol, iso-octane (99.5%), acetic acid (99%), Carbon disulfide (CS2, 99.9% purity), and formic acid (95%), hydrochloric acid (35%), diethylene glycol (DEG), Sodium sulphate (Na 2 SO 4 ), ammonium formate (Merck India Ltd., Mumbai), and primary secondary amine (PSA) (Agilent Technologies, Santa Clara, CA, USA). The individual stock solutions of certified pesticide standards (10 ± 0.10 mg) were prepared in methanol (10 ± 0.10 g) and stored at -20°C for fungicides, insecticides, and plant growth regulators. Thereafter, intermediate (10 µg/mL) and working (1 µg/mL) standard solutions for matrix-matched and solvent standard calibrations were made through serial dilution in methanol. CS₂ solution (stock) was prepared for carbon disulfide for di-thiocarbamates by pipetting 10 µL of CS₂ standard into 10 mL of iso-octane. Then, working solutions (1 and 0.1 mg/kg) were obtained by the serial dilution method with iso-octane solvent. A separate thiram stock solution was prepared by mixing 10 µL of the standard in C 4 H 8 O 2 (ethyl acetate). After that, a 1 mg/kg working solution of thiram was then prepared by serial dilution. The reaction mixture for dithiocarbamates was prepared by dissolving 30 g of stannous (II) chloride in 1000 mL of 35% HCL, followed by the gradual addition of 1000 mL of water and sonication for 2 minutes Sample Preparation and Analytical Instruments For the detection of residues of fungicide and insecticide, a homogenized grape sample (10.00 ± 0.10 g) was weighed into a centrifuge tube (50 mL). The 10 mL of C 4 H 8 O 2 was then added to the homogenized grape sample, and thoroughly mixed at 15,000 rpm for 1 minute. Anhydrous sodium sulfate (10 g) was added next, and the homogenization was repeated under the same conditions. The sample extract was centrifuged at 5000 rpm for 5 min and subsequently processed by LC-MS/MS (Shimadzu UFLC XR system coupled with mass spectrometer (AB Sciex API 4000 QqQLIT) and GC-MS/MS (Shimadzu TQ8040 GC-MS/MS system (Shimadzu Corporation, Japan) armed with an Rxi®-5Sil MS column (Restek Corporation, USA). Similarly, for detecting residues of ethephon, the homogenized sample (10.00 ± 0.10 g) was accurately weighed, and 20 mL of acidified methanol (1% formic acid) was added. Further, the mixture was mixed for 2 min and centrifuged for 5 min at 5000 rpm. Then, 500 µL of the separated aqueous layer was combined with 500 µL of acetonitrile for analysis of ethephon residues. Extraction of the di-thiocarbamate residue analysis, the grape berries (±25.01 g) were placed in a glass bottle with having 250 ml capacity. After that, a 75 mL recipe (30 g Stannous (II) chloride in 1000 mL HCL + 1000 mL water) was added, followed by 25 mL of iso-octane, and the bottle was capped quickly. After that, the bottle was incubated in a water bath at 80°C and shaken intermittently every 20 minutes for 1 hr. Then the bottle was dipped in ice-cold water to cool it down to <20 °C. 1 mL of the upper iso-octane layer aliquot was added to a new tube and subjected to centrifugation for 5 min at 10 °C. After injecting the aliquot into the GC, di-thiocarbamate residues were calculated as CS 2 using GC/MS. Optimized parameters for individual pesticides, both for GC-MS/MS and LC-MS/MS, are provided (Supplementary Table 1). In both the ionization modes (positive and negative), mass spectrometric identification was carried out. Information procurement and estimation for LC-MS/MS were performed using Analyst software (v1.7.1). However, for di-thiocarbamates, mass spectrometry was operated in SIM mode with a temperature of 40-290°C (Supplementary Table 2). Argon was used as the collision gas, and mass acquisition began at 4 minutes. Data acquisition and processing GC-MS/MS was performed using LabSolutions® software (Version 4.50, Japan). The analytical method SANTE/ 11312/2021 was followed (SANTE, 2021). Statistical Analysis The field observations on treatment were repeated three times and recorded. The recorded data was analyzed for the calculation of analysis of variance (ANOVA) by randomized block design with the help of OPSTAT, an online tool. The initial recorded data was converted to arcsine values and used for ANOVA. The standard error of mean, standard error of deviations, and critical difference (C.D.) values were estimated. The level of significance is indicated at the bottom of the tables. RESULTS Morphological Identification of Fungal and Bacterial Endophytes The 13 endophytic isolates were successfully purified, which originated from berries, stems, roots, and leaves of the grapevine cv. Manik Chaman, dogridge, Muscut, Sauvignon Blanc, and Vitis rotundifolia (Fig. 1 and Supplementary Table 3). Based on the morphological key characteristics, fungal endophytes viz ., CSBY-2, CSBY-4, CSBY-8, MCBY-2, and DRRS-1 were identified as Trichoderma sp. All the fungal endophytes produced distinctive green to olive-green colonies, powdery texture, and rapid growth on the PDA medium. Based on the microscopic observations endophytic Trichoderma isolates showed septate hyphae, branched conidiophores, and globose conidia. The four EB strains viz ., SB5, SB2, CS2, and RF1 were earlier characterized (Holkar et al., 2024 ), whereas three EB strains viz ., RT7, RB1, and GB2 were characterized in the present study. The EB strains were Gram’s Gram-positive, mucoid colonies, with convex elevations having circular to irregular shapes and white-colored colonies. Molecular Identification of Endophytic Microbes Based on ITS sequence information, the endophytic Trichoderma isolates viz ., CSBY2, CSBY8, and MCBY2 were identified as Trichoderma asperellum. In contrast, CSBY4, DRRS1, and MCBY1 were identified as T. asperelloides , strains viz ., Sauvignon Blanc − 5, -2, Crimson Seedless − 2, and Rotundifolia − 1 were recognized as Bacillus subtilis , whereas RT7, RB1, and GB2 strains were recognized as B. mojavensis , and B. licheniformis , respectively (Supplementary Table 3 and Fig. 2 ). Sequence information of all the isolates was deposited in the GenBank and received the accession numbers for each isolate. Effects of Endophytic Trichoderma and Bacillus Species on Physical Parameters of Grapes The efficacy of various Trichoderma and Bacillus -based bio-formulations in improving the physical parameters of cv. Sarita Seedless was evaluated during the 2022-23 and 2023-24 crop seasons. T1 to T16 treatments with untreated control (UTC) were followed. During the 2022–2023 crop growing season, treatments showed positive results in enhancing specific physical traits. Results revealed that T4 ( Bacillus licheniformis strain RB1) treatment improved average bunch weight (450.4 g), pH (3.27), and yield (13.962 kg/vine); T6 ( Bacillus mojavensis strain RT7) increased 50-berry weight (224.8 g), berry length (2.75 mm), and berry diameter (1.56 mm); T10 ( Cytospora species MCBY1 isolate) increased the number of berries per bunch (95); T15 ( Bacillus subtilis strain SB2 + Cytospora species MCBY1 isolate) enhanced skin thickness (0.27 mm) and rachis length (238.88 mm); T8 ( Trichoderma viride isolate DRRS1 ) improved pedicel length (13.54 mm) and single bunch weight (152.26 g); T1 ( Bacillus spp. strain GB2) increased TSS (21.4), and T13 ( T. viride isolate DRRS1 + B. subtilis strain SB2) reduced acidity (0.87) as described in (Supplementary Table 4 and Fig. 3 ). During the 2023–2024 crop-growing season, different treatments showed improved physical parameters. T5 ( B. subtilis strain SB2) increased average bunch weight (556.67 g) and TSS (23.35); T3 ( T. asperellum isolate MCBY2) enhanced 50-berry weight (249 g) and pedicel length (14.71 mm); T12 ( T. asperellum isolate MCBY2 + B. subtilis strain SB2) improved the number of berries per bunch (114); T7 ( T. asperelloides isolate CSBY4 + B. subtilis strain SB5) increased skin thickness (0.38 mm); T15 ( T. asperelloides isolate CSBY4 + T. viride isolate DRRS1 + T. asperellum isolate MCBY2 + B. subtilis , SB5 + B. subtilis , SB2 + B. subtilis strain CS2) lengthened rachis length (249.78 mm) and raised acidity (2.6); T16 (positive control with sulphur) improved single bunch weight (184.89 g); T6 increased berry length (2.7 mm); T9 ( T. asperellum isolate MCBY2 + B. subtilis strain SB5) enhanced berry diameter (1.79 mm) and pH (3.96); and T1 ( T. asperelloides isolate CSBY4) increased yield (14.377 kg/vine), as detailed in (Supplementary Table 5 and Fig. 3 ). During both the crop growing season, bioformulations of RB1, CSBY4, MCBY2, SB5 and DRRS1 showed promising results in all the physical parameters. Effects of Endophytic Bioformulations on Biochemical Parameters of Grapes Biochemical observations from grape samples of cv. Sarita Seedless, collected during two consecutive crop growing seasons i.e. , 2022–2023 and 2023–2024 at ICAR-NRCG, Pune experimental farm. Multiple biochemical traits including, phenol, tannin, reducing sugar, protein, proline, carbohydrate, and antioxidants across 16 different treatments (T1–T16) and untreated control (UTC) were compared. The data indicated that phenol (mg/g) content ranged from 0.45 (T1: GB2) to 0.63 (T10: MCBY1) and 0.14 (T15: CSBY4 + DRRS1 + MCBY2 + SB5 + SB2 + CS2) to 0.86 (T2: DRRS1) during the years 2022-23 and 2023-24, respectively (Supplementary Table 6). Tannin levels ranged from 0.57 (T1) to 0.82 (T10) and 0.51 (T13) to 0.89 (T10) during the same year. Reducing sugar content was recorded higher in 2022-23 ranging from 19.03 (T4: RB1) to 59.1 (T3: SB2). It was recorded lowest during the 2023-24 crop season, ranging from 14.87 (T4: SB5) to 43.75 (T9: MCBY2 + SB5). In the year, 2022-23 protein level ranged from 1.26 (T16: PC with Sulphur) to 1.84 (T2: SB5), and in 2023-24 it was 0.74 (UTC) to 1.97 (T2: DRRS1). Proline levels varied significantly, ranging from 1.36 (T10: MCBY1) to 5.59 (T5: RF1) and 3.45 (UTC) to 5.14 (T12: MCBY2 + SB2) during 2022-23 and 2023-24, respectively (Supplementary Table 6). Effect of Endophytic Trichoderma and Bacillus Isolates on Shelf-Life of Grapes Benefits of bioformulations on the lifespan of grape berries were carried out based on loss in initial weight during five days at room temperature, 28 ± 2 o C, as indicated in Supplementary Table 7. It was noted that T6 (RT7) had the lowest weight loss in 2022–2023 at 2.17%, but this increased significantly to 5.31% in 2023–2024, revealing one of the better-performing treatments in improving the shelf-life of grape berries. T5 (RF1) also demonstrated good performance, with 3.66% weight loss in 2022–2023, to 4.93% (T5: SB2) in 2023–2024, indicating a relatively stable shelf-life across both years. On the other hand, T15 exhibited the highest weight loss in 2023–2024 at 8.43%, compared to 4.30% in 2022–2023. The UTC also showed a significant increase in weight loss from 5.75–6.17% in 2022-23 and 2023-24. The treatments like T4, T7, T8, and T10 showed a moderate increase in weight loss between the two seasons. For instance, T4 had 5.09% weight loss in 2022–2023, which increased to 5.69% in 2023–2024 (Supplementary Table 7). The treatment with DRRS1 + SB2 provided the minimum weight loss (4.42%) in 2022-23 whereas, SB2 alone in 2023-24 showed 4.93% weight loss, therefore the application of SB2 was found effective in improving the shelf-life with minimum weight loss. The standard error ranged from 51.99 to 79.564 for initial weight and from 51.192 to 72.542 for final weight, with coefficients of variation (CV%) between 22.065% and 26.645%. Statistical significance for weight loss was observed at a 5% level of significance (p = 0.00542 for 2022–2023), highlighting the effectiveness of specific treatments, particularly T6, in reducing weight loss and enhancing the post-harvest shelf-life of grapes (Supplementary Table 7 and Fig. 4 ). Bio-Efficacy of Endophytes against Powdery Mildew Disease of Grapes During both the crop growing seasons 2022-23 and 2023-24, a total of 13 bioformulations were field evaluated against powdery mildew. Based on the in vivo evaluations during 2022-23, only three fungal and three bacterial bio-formulations and their combinations were evaluated during 2023-24. The per cent disease index (PDI) during 2022-23 ranged from 25.18–68.83% on leaves and 23.82–67.19% on bunches (Table 1 ). Likewise, during 2023-24, PDI ranged from 27.99–66.83% on leaves and 21.46–66.59% on bunches (Table 2 ). All 13 bioformulations showed per cent disease control (PDC) as compared to UTC. The PDC during the 2022-23 season ranged from 12.49–63.41% and 44.5–64.56% on leaves and bunches, respectively (Table 1 and Fig. 5 ). Likewise, the PDC during the 2023-24 season ranged from 26.0–63.18% and 44.97–67.7% on leaves and bunches, respectively (Table 2 and Fig. 5 ). During 2022-23 crop season, five bioformulations viz., T1 (GB2), T3 (SB2), T5 (RF1), T6 (RT7) and T15 (SB2 + MCBY1) showed maximum PDC ranged from 42.55–63.41% and 49.73–64.56% on leaves and berries, respectively (Table 1 and Fig. 5 ). Similarly, during 2023-24 crop season, five treatments viz ., T3 (MCBY2), T5 (SB2), T13 (CSBY4 + DRRS1 + MCBY2), T14 (SB2 + SB5 + CS2) and T15 (CSBY4 + DRRS1 + MCBY2 + SB2 + SB5 + CS2) showed to be potential treatments in PDC ranged from 58.11–63.18% and 48.60–67.7% on leaves and bunches, respectively (Table 2 and Fig. 5 ). During both the crop growing seasons, MCBY2 (60.93% and 67.7%), SB2 (61.57% and 64.56%) isolates performed to be the best among all the bioformulations field evaluated. Moreover, RF1 (63.41%), GB2 (61.04%), and combinations of T. viride isolate DRRS1, T. asperellum isolate MCBY2 and T. asperelloides isolate CSBY4 (63.18%) showed promising results in managing the disease on leaves (Table 1 , 2 and Fig. 5 ). All the formulations were tested for their compatibility with the chemical fungicides, it was found to be highly compatible with propineb 70 WP, and a combination of 5% kasugamycin and 45% WP copper oxychloride, and 18% kresoxim methyl with 54% mancozeb. Whereas, the formulations were moderately compatible with copper oxychloride 50 WP, 8.3% azoxystrobin and 66.7% WG mancozeb, and 47.15% copper sulphate with 30% mancozeb. These formulations were not compatible with the chemical fungicides, viz ., fluopyram 200 + tebuconazole 200 SC, carbendazim 50 WP, thiophanate methyl 70 WP, carbendazim 12% + mancozeb 63% WP. The solid-state formulations were stable for six months with a spore count of 10 11 /ml, after six months spore count was reduced to 10 10 / ml under refrigerated conditions. Trichoderma solid-state formulations retained their viability for 12 months with a drop in one log. Whereas, the bacterial liquid state formulations were found to be viable for six months when stored under refrigerated conditions. Table 1 Field evaluation of Trichoderma and Bacillus formulations during the 2022-23 crop growing season in grapevine cv. Sarita Seedless at experimental farm of ICAR-NRCG, Pune, Maharashtra, India. Treatments PDI on leaves (%) PDC PDI on Bunches (%) PDC Before spray After 1st spray After 2nd spray After 3rd spray After 4th spray (%) Before spray After 1st spray After 2nd spray After 3rd spray After 4th spray (%) T1 5.76 (13.83) 10.21 (18.41) 16.04 (22.71) 21.11 (26.06) 26.81 (33.61) 61.04% 4.91 (9.18) 11.45 (21.17) 20.44 (26.11) 26.17 (30.21) 30.16 (36.16) 55.12% T2 7.01 (14.31) 10.96 (18.74) 17.48 (25.81) 24.00 (31.18) 27.76 (33.21) 59.66% 4.22 (11.95) 13.32 (21.56) 20.68 (24.31) 24.42 (28.16) 29.44 (33.98) 56.18% T3 7.77 (14.94) 10.32 (17.24) 18.21 (23.86) 24.60 (31.18) 26.44 (33.36) 61.57% 4.08 (11.23) 14.49 (21.19) 16.20 (26.31) 20.16 (34.32) 23.82 (38.69) 64.56% T4 5.01 (12.92) 11.24 (18.42) 19.6 (23.14) 24.16 (28.74) 28.96 (32.58) 57.91% 5.10 (11.67) 15.18 (23.34) 22.12 (28.82) 29.53 (34.30) 35.91 (37.48) 46.55% T5 5.96 (13.40) 10.13 (16.13) 17.31 (24.72) 22.13 (30.66) 25.18 (34.41) 63.41% 4.50 (12.18) 16.76 (23.09) 22.11 (28.60) 31.17 (33.88) 33.78 (37.96) 49.73% T6 6.86 (16.02) 14.47 (21.11) 23.43 (31.66) 28.18 (34.71) 39.54 (37.22) 42.55% 5.80 (13.90) 12.38 (20.52) 19.20 (25.94) 23.20 (28.73) 27.48 (32.83) 59.11% T7 4.91 (12.52) 9.21 (18.48) 16.00 (22.84) 22.63 (29.42) 28.30 (31.33) 58.89% 3.92 (11.40) 14.28 (22.08) 21.46 (27.50) 27.76 (32.43) 32.16 (34.33) 52.14% T8 6.4 (14.94) 21.38 (28.01) 34.11 (36.89) 53.51 (46.44) 60.23 (51.00) 12.49% 3.64 (10.89) 13.61 (21.58) 25.16 (30.04) 31.11 (34.42) 34.90 (39.66) 48.07% T9 5.98 (14.01) 12.12 (20.54) 21.34 (27.41) 26.14 (30.67) 29.76 (32.14) 56.75% 4.22 (11.78) 12.26 (20.42) 20.84 (27.11) 26.16 (30.72) 30.19 (35.14) 55.06% T10 5.05 (13.53) 16.22 (23.68) 25.14 (30.20) 32.00 (35.04) 37.19 (38.25) 45.96% 3.26 (10.38) 12.87 (20.96) 20.21 (26.62) 26.60 (30.95) 32.81 (35.87) 51.17% T11 8.89 (16.19) 17.51 (24.95) 23.27 (28.68) 30.18 (31.19) 33.65 (37.01) 51.07% 3.55 (10.81) 12.91 (20.98) 19.20 (25.89) 24.27 (29.44) 29.58 (34.11) 55.88% T12 10.73 (19.18) 16.48 (23.80) 23.51 (28.36) 27.44 (31.41) 30.04 (35.92) 55.85% 3.45 (11.00) 14.28 (22.15) 24.16 (29.38) 31.59 (34.15) 35.17 (37.13) 47.61% T13 6.61 (16.00) 15.88 (22.58) 21.62 (32.03) 26.81 (30.71) 33.07 (37.00 51.96% 4.04 (11.96) 13.71 (21.13) 21.10 (26.51) 26.55 (30.29) 30.71 (34.81) 54.30% T14 10.04 (19.46) 21.68 (28.00) 28.64 (32.03) 37.46 (38.82) 39.56 (41.12) 42.68% 3.90 (11.29) 11.77 (19.14) 24.70 (28.24) 28.16 (35.87) 33.22 (39.04) 50.56% T15 7.04 (16.13) 15.60 (23.24) 20.15 (26.71) 26.97 (31.23) 30.86 (38.00) 55.13% 3.06 (10.07) 13.15 (21.21) 19.17 (25.89) 23.94 (29.84) 27.41 (32.63) 59.2% T16 12.80 (20.17) 16.16 (23.14) 21.19 (27.37) 26.19 (30.18) 32.48 (33.61) 52.80% 3.35 (10.52) 12.26 (20.41) 17.12 (25.92) 27.03 (30.58) 37.26 (40.40) 44.5% UTC 12.65 (19.14) 27.13 (31.76) 43.12 (38.55) 66.35 (54.61) 68.83 (58.66) 4.51 (12.13) 25.22 (30.95) 36.23 (37.31) 53.10 (47.16) 67.19 (69.98) SE ± 0.84 1.36 1.28 1.42 1.54 0.71 1.26 1.54 1.81 1.96 CD 5% NS 5 4.66 4.94 5.62 - NS 3.68 4.49 5.64 5.72 - CV (%) 9.81 9.94 9.18 8.44 8.28 - 12.19 10.88 10.95 10.83 10.96 - Level of significance at p-value ≤ 0.05 except before the spray Table 2 Field evaluation of Trichoderma and Bacillus formulations during the 2023-24 crop growing season (pooled mean of two seasons of respective treatments) in grapevine cv. Sarita Seedless at experimental farm of ICAR-NRCG, Pune, Maharashtra, India Treatment PDI on leaves (%) PDC PDI on Bunches (%) PDC Before spray After 1st spray After 2nd spray After 3rd spray After 4th spray (%) Before spray After 1st spray After 2nd spray After 3rd spray After 4th spray (%) T1 6.84 (14.93) 11.2 (19.59) 18.23 (24.66) 23.61 (28.39) 28.11 (33.61) 57.92% 4.04 (9.48) 12.89 (20.27) 21.19 (27.31) 26.17 (30.21) 31.46 (36.18) 52.07% T2 7.06 (14.32) 11.56 (19.22) 18.12 (26.45) 24.32 (31.22) 28.68 (34.18) 57.09% 4.21 (11.75) 13.92 (21.82) 19.73 (26.31) 24.72 (29.76) 29.36 (33.91) 55.87% T3 7.81 (15.03) 9.62 (16.98) 17.51 (24.91) 22.16 (29.48) 26.09 (33.16) 60.93% 3.17 (10.23) 13.49 (21.49) 14.20 (27.30) 17.16 (31.32) 21.46 (38.69) 67.7% T4 5.97 (13.24) 12.65 (19.93) 19.6 (23.14) 24.16 (28.74) 28.96 (32.58) 56.65% 4.14 10.67 14.48 (22.33) 21.92 (27.82) 28.76 (32.30) 35.44 (37.26) 46.07% T5 6.35 (14.48) 12.24 (18.92) 19.54 (26.56) 24.87 (32.43) 27.99 (37.14) 58.11% 3.70 (11.08) 14.96 (22.71) 23.17 (28.70) 31.17 (33.88) 34.71 (38.07) 44.97% T6 7.93 (16.33) 18.7 (25.63) 27.84 (31.66) 36.54 (36.98) 42.76 (43.39) 36.03% 5.80 (13.90) 12.38 (20.52) 19.20 (25.94) 23.20 (28.73) 27.33 (32.83) 63.00% T7 5.77 (13.67) 10.86 (19.01) 16.09 (22.87) 23.91 (30.46) 29.41 (34.89) 56.00% 3.92 (11.40) 14.28 (22.08) 21.46 (27.50) 28.88 (32.43) 33.96 (35.12) 52.71% T8 6.7 (14.96) 22.42 (28.21) 36.62 (37.2) 44.46 (49.89) 49.10 (53.14) 26.0% 3.64 (10.89) 13.61 (21.58) 25.16 (30.04) 32.01 (34.42) 36.20 (39.66) 45.67% T9 6.62 (14.81) 13.42 (21.38) 20.22 (26.60) 26.14 (30.67) 30.58 (32.74) 54.25% 4.22 (11.78) 12.26 (20.42) 20.84 (27.11) 26.16 (30.72) 31.30 (35.14) 52.97% T10 6.09 (14.23) 16.22 (23.68) 26.10 (30.68) 33.87 (35.54) 38.90 (39.23) 41.74% 3.26 (10.38) 12.87 (20.96) 20.21 (26.62) 26.60 (30.95) 31.59 (34.98) 52.56% T11 9.21 (17.59) 18.68 (25.55) 23.07 (28.62) 29.30 (32.69) 34.38 (37.33) 48.54% 3.55 (10.81) 12.91 (20.98) 19.20 (25.89) 24.27 (29.44) 29.40 (34.11) 61.00% T12 10.78 (19.15) 16.22 (23.73) 22.46 (28.25) 27.10 (31.32) 31.42 (36.04) 53.02% 3.67 (11.00) 14.28 (22.15) 24.16 (29.38) 31.59 (34.15) 36.27 (37.13) 46.39% T13 9.77 (18.16) 12.25 (27.42) 16.22 (32.69) 20.40 (38.86) 24.61 (42.72) 63.18% 3.64 (10.96) 12.70 (20.83) 20.00 (26.51) 25.55 (30.29) 29.97 (33.81) 59.63% T14 7.91 (16.29) 16.05 (23.58) 21.64 (27.68) 27.72 (31.73) 34.12 (38.06) 59.66% 3.90 (11.29) 11.77 (19.14) 24.70 (28.24) 29.95 (35.87) 34.22 (39.04) 48.60% T15 7.86 (16.24) 15.50 (23.14) 21.35 (27.47) 26.97 (31.23) 31.84 (38.41) 62.41% 3.06 (10.07) 13.15 (21.21) 19.17 (25.89) 24.94 (29.84) 29.46 (32.63) 58.53% T16 10.80 (19.17) 16.12 (23.64) 21.19 (27.37) 25.19 (30.08) 34.31 (32.64) 48.66% 3.35 (10.52) 12.26 (20.41) 19.22 (25.92) 26.03 (30.58) 36.03 (39.40) 45.89% UTC 11.65 (19.44) 26.13 (31.06) 40.62 (38) 63.18 (52.66) 66.83 (56.15) - 3.97 (11.39) 24.12 (29.35) 35.23 (36.36) 52.70 (46.56) 66.59 (69.98) - SE ± 0.87 1.28 1.18 1.38 1.46 - 0.67 1.19 1.36 1.68 1.74 - CD 5% NS 4 4.28 4.65 4.91 - NS 3.45 4.21 5.31 5.48 CV (%) 9.68 9.82 9.02 8.12 8.06 - 12.4 10.34 10.13 10.62 10.88 - All the treatments were significant at p-value ≤ 0.05 except before the spray Residue Analysis Residue analysis of the Sarita Seedless grape variety under various treatments was conducted to determine the presence of multiple pesticides and their compliance with maximum residue limits (MRLs). Across all samples, residues of ethephon were observed in the range 0.010 to 0.363 mg/kg, remaining were below the MRL of 1.00 mg/kg. Residues of Di-thiocarbamates together assessed as CS 2, varied from 0.191 to 0.639 mg/kg, in the allowed limit of 5.00 mg/kg. In buprofezin, residues were detected in most treatments, values ranged from 0.015 to 0.486 mg/kg, exceeding the allowable limit of 0.01 mg/kg in several cases (Fig. 6 ). Similarly, clothianidin showed 0.044 to 0.092 mg/kg, remaining below its limit of 0.70 mg/kg. The fungicide dimethomorph values showed 0.106 to 0.404 mg/kg, all within the allowable MRL of 3.00 mg/kg. The hexaconazole residual limit of 0.01 mg/kg was frequently detected, ranging between 0.010 and 0.022 mg/kg, often over and above the demarcated limit. For mandipropamid, residues limits were observed as 0.299 to 0.696 mg/kg, staying within the 2.00 mg/kg threshold. The fungicide metrafenone was present in concentrations between 0.392 and 0.654 mg/kg, below its MRL of 7.00 mg/kg in all samples (Fig. 6 ). Tebuconazole residues ranged from 0.015 to 0.352 mg/kg, within the MRL of 0.50 mg/kg, except in untreated controls. Additionally, 4-bromo-2-chlorophenol was detected in rootstock-treated samples at 0.183 mg/kg, above the standard limit of 0.01 mg/kg. Overall, most pesticide residues remained within their respective MRLs, indicating compliance with safety standards, except for buprofezin, hexaconazole, and 4-bromo-2-chlorophenol, which were found above permissible limits in specific treatments (Fig. 6 ). The validated method demonstrated satisfactory linearity in the grape matrix, with correlation coefficients (r²) > 0.990. As per the result, the limit of quantification (LOQs) ranged from 0.004–0.009 mg/kg, below the EU-established MRL for grapes. However, mean recoveries of selected pesticides were 78.40–112.31%, with a percentage relative standard deviation (RSD) between 1.28–6.85%, whereas, the matrix effects ranged from − 7.71–23.2%, indicating minimal interference (Table 3 ). Table 3 Residue analysis method validation recovery (%), and matrix effect (%) for selected pesticides in grape Sr. No. Name of the compound Instrument used for analysis Mean % recovery at 0.010 mg/kg (n = 6) % RSD Mean % recovery at 0.050 mg/kg (n = 6) % RSD Mean % recovery at 0.100 mg/kg (n = 6) % RSD ME (%) at 0.010 mg/kg LOQ at 0.010 mg/kg 1 4-Bromo 2- chlorophenol GC-MS/MS 84.20 2.75 87.60 3.45 95.10 1.60 19.30 0.008 2 Buprofezin LC-MS/MS 92.10 5.21 98.40 2.85 105.30 2.40 -10.14 0.004 3 Clothianidin LC-MS/MS 81.20 4.30 89.60 3.52 97.45 2.11 -16.42 0.006 4 Dimethomorph LC-MS/MS 88.35 3.25 85.70 3.05 95.72 2.65 -5.62 0.005 5 Dithiocarbamates (Mancozeb, Maneb, Propineb, Metiram, Thiram, Zineb and Ziram collectively estimated as CS2 GC-MS/MS 78.40 6.85 84.30 5.72 92.40 4.28 23.20 0.009 6 Ethephon LC-MS/MS 81.70 5.25 88.48 6.51 102.37 3.45 -31.22 0.009 7 Etrimfos LC-MS/MS 79.50 7.21 83.70 6.04 89.40 4.52 -28.45 0.008 8 Fluchloralin GC-MS/MS 83.85 6.15 80.65 5.48 91.18 3.21 -21.64 0.004 9 Hexaconazole LC-MS/MS 95.10 3.75 105.36 2.16 110.20 1.62 -9.35 0.006 10 Mandipropamid LC-MS/MS 91.54 4.15 96.31 3.77 103.71 2.33 -13.47 0.005 11 Metrafenone LC-MS/MS 94.22 3.71 99.82 2.39 106.55 1.76 -11.30 0.007 12 Tebuconazole LC-MS/MS 98.85 2.64 107.51 2.15 112.31 1.28 -7.71 0.005 RSD: Relative standard deviation, ME: Matrix effect, LOQ: Limit of quantification DISCUSSION The present study aimed to isolate the endophytic microbes, purify, characterize, develop their formulations, and evaluate their bio-efficacy against powdery mildew disease under field conditions in the grapevine cv. Sarita Seedless is a black-coloured table grape variety largely cultivated in the state of Maharashtra, India. The application of fungicides is limited for producing EU-MRL residue-compliant grapes. Hence, the aim was to manage disease by reducing the reliance on chemical fungicides and improving the quality and yield. The table and export grapes require minimal pesticide residues to meet consumer expectations, and the final harvest with the residual effect of chemicals is considered undesirable (Dumitriu et al. 2021 ; Warneke et al. 2022 ). To align with the healthy produce, many vineyards adopt "zero pesticides" approach, thereby encouraging organic and natural practices (Roskaric et al. 2023 ). This investigation assessed the efficient endophytic microbes to effectively manage powdery mildew disease. The impact of microbial inoculants on disease development was compared to sulphur-treated (positive control) and untreated plants. The chemical fungicides detected in the present study were applied before the flowering stage of the crop. A comprehensive analysis was conducted to evaluate the treatment effects of PDI on foliage and berries, shelf-life, and loss in weight. Biochemical analyses of endophyte-treated grape samples included measurements of phenol, tannin, reducing sugars, proteins, proline, carbohydrates, and antioxidants. During both the crop seasons 2022-23 and 2023-24, all the isolates tested against grapevine showed excellent results on physical parameters of grapevine, including average berry weight, rachis length, total soluble solids (TSS), pH, berry length, and diameter, and other characteristics. In addition to this, the biochemical analysis of grapevine samples was estimated during both the years 2022-23 and 2023-24, which showed a positive impact on reducing sugar, tannin, protein, carbohydrates, and antioxidant properties of berries harvested from experimental plots. These findings were supported by the previous work evidenced that Trichoderma application enhanced beneficial effects on TSS, pH, and acidity of grapevine crops (Csoto et al. 2022 ; Carro-Huerga et al. 2023 ). Moreover, Trichoderma spp. not only increases the nutritional properties of the grapevine but it increases the accumulation of sugar in grapevine berries (Csoto et al. 2022 ). Additionally, plants treated with endophytes consistently outperformed untreated controls across all experimental sites, and distinct reduction in the spread of disease in the treated vineyards. These findings were in accordance with the earlier reports on percent disease control with enhanced yield and quality (Bruisson et al. 2019 ; Malviya et al. 2022 ; Nasehi et al. 2023 ). Efficacy of the fungal and bacterial endophytes could be achieved by various mechanisms, as evidenced from the recent research (Fadiji et al. 2020; Malviya et al. 2020 , 2022 ), that led to inhibition of E. necator in grapevine. Successful foliar sprays by these biocontrol agents indicate the strong rhizosphere and phyllosphere competence, a crucial factor for an effective biocontrol strategy (Santos et al. 2021 ). Moreover, the endophytic Trichoderma asperellum and T. asprelloides could have supplemented the systemic resistance (ISR) in grapes. This mechanism protects against phytopathogens and environmental stressors (El-Sharkawy et al. 2018 ; Pacifico et al. 2019 ). Previous findings supported this investigation by Sawant et al. ( 2020 ), in vivo applications of rhizospheric Trichoderma developed ISR in grapes against the same pathogen. Previously, the efficiency of Bacillus spp. for powdery mildew has been extensively studied (Maachia et al. 2015 ; Boiu-Sicuia et al. 2023 ; Saha et al. 2023 ; Holkar et al. 2024 ). The EB strains viz ., SB5, RF1, SB2, and CS2 used in the present study were earlier assessed under laboratory conditions for direct and indirect antagonism for C. gloeosporioides , the incitant of anthracnose in grapes in India (Holkar et al. 2024 ) and found promising results. Moreover, field evaluations of these bioformulations for anthracnose disease are also in process. In the recent past, many research investigations have confirmed that the application of microbial formulations improved the nutritive quality parameters of grapevine and the health of plants simultaneously (Lombardi et al. 2020 ; Verdenal et al. 2021 ; Bettenfeld et al. 2022 ). The investigation from this research supported quality parameters, but did not study the specific metabolites in fruits. The application of sulphur 80% WDG is an effective fungicidal strategy for powdery mildew disease management and to improve grapevine yield (Warneke et al. 2022 ). Sulphur fungicide has protective properties rather than curative action. Therefore, during the present investigation, sulphur 80% WDG @ 2 g/L was used as a positive control. Sulphur strongly invades Erysiphe necator and prevents vineyards (Sellitto et al. 2021 ). In the vines treated with endophytic formulations, sulphur, and other fungicides were not used till the final harvest of the crop, except for the positive control. Therefore, the earlier bio-formulations of rhizospheric microbes were found efficient by integrating their application in combination with sulphur for enhanced disease management (Malviya et al. 2022 ). This study significantly reduced disease on foliage and berries with improvement in post-harvest life when endophytic microbes’ bio formulation was applied to the grapevine than the PC (S) and SDW treatments. Residue analysis of cv. Sarita Seedless grapevine berries revealed that ethephon, di-thiocarbamates, clothianidin, dimethomorph, mandipropamid, metrafenone, and tebuconazole were all within the EU-MRL permissible limits. In the recent past, a strain of Enterobacter spp. originated from the Morus alba . Significantly reduced residues of Thiamethoxam in Chinese cabbage (Wang et al. 2020 ). Five endophytic bacterial strains having plant growth-promoting activities degraded > 90% of chlorpyrifos in rice plants and grains (Feng et al. 2017 ). Moreover, in the recent past, Kumar et al. (2020) studied the biodiversity of endophytic microbial communities, including Bacillus and Trichoderma species were found to degrade carbamate, organochlorine, organophosphate, and pyrethroids. In India, many endophytic microbes were isolated and found to have pesticide residue degradation ability in different crops and heavy metal detoxification (Mukherjee et al. 2021 ; Hazra et al. 2023 ; Shahid et al. 2023 ; Sharma et al. 2024 ). Chemical residues were recently estimated from grape leaves infected by fungal diseases (Ailer et al. 2024 ). In India, scant data are reported on pesticide residue management through endophytic microbes. Therefore, in the present study novel endophytic microorganisms were characterized for the management of powdery mildew disease and pesticide residues in the final harvest. The present formulations viz ., GB1, RF1, SB2, MCBY2, CSBY4, DRRS, and SB5 were found efficient in managing the disease and enhancing physiological and biochemical characteristics of the grape berries. Additionally, application of these bioformulations reduced the pesticide residues on the berry surface below the EU-MRL limits. This indicated that these grapevine endophytic microbes have multifaceted potential in grapes. Further, among these three bioformulations, viz ., MCBY2, DRRS, and SB5, were accepted for the multi-locational testing (MLTs). This information will be helpful for their registrations and legal usage in grapes. Moreover, combining all Trichoderma bioformulations and Bacillus provided significant effects during 2023-24. This indicated that a consortium of Trichoderma isolates is needed for devising an efficient disease management strategy in grapevine. CONCLUSION The present study demonstrated the impact of endophytic microbial formulations of Trichoderma and Bacillus species in managing powdery mildew disease in grapevines while improving the physical, biochemical, and shelf-life parameters of grape berries. Residue analysis indicated compliance with EU-MRLs for most of the pesticides, ensuring safety and suitability for consumption and export. The study underscores the potential of integrating endophytic microbes into sustainable farming practices, offering an eco-friendly, safe and optional strategy for chemicals in disease management, especially when fungicide application at berry development stage is not possible. Declarations The authors declare that they have no conflict of interest in the publication. SKH: conceived ideas and formulated the experiments, drafted and reviewed the manuscript, SCN: isolated, characterized, and evaluated the bioformulations of endophytes under field conditions and prepared tables, SBB: analysed sequence information and constructed phylogenetic trees, SJP: prepared the figures and reviewed the manuscript, HNM: reviewed and drafted the manuscript, BST: residue analysis of the grape samples, SDG: Statistical analysis, NAD: reviewed and drafted the manuscript, KB: critically reviewed the manuscript. Conflict of Interest Authors declare that they have no conflict of interest. Accession Numbers OQ407830, OQ402731, OQ781200, OQ503170, OQ784170, OM280451, OL778931, OM280455, OM341599, OL797982, ON063017, OQ473591 Acknowledgements All the authors highly acknowledge the support provided by the Director, ICAR-National Research Centre, Pune to carry out the proposed research work. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Ailer S, Benesova L, Baron M, Galovicova L (2024) Monitoring of pesticide residues in grapevine leaves under variant fungal disease management. 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We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6602877","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":456946655,"identity":"af3ab548-d964-4c4e-bdcf-7bfd167d7d79","order_by":0,"name":"Somnath K. 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Taynath","email":"","orcid":"","institution":"Indian Council of Agricultural Research-National Research Centre for Grapes","correspondingAuthor":false,"prefix":"","firstName":"Bharat","middleName":"S.","lastName":"Taynath","suffix":""},{"id":456946661,"identity":"df48a1d2-80e5-4194-8efb-49ebea9e04ce","order_by":6,"name":"Sudarshan D. Gat","email":"","orcid":"","institution":"Indian Council of Agricultural Research-National Research Centre for Grapes","correspondingAuthor":false,"prefix":"","firstName":"Sudarshan","middleName":"D.","lastName":"Gat","suffix":""},{"id":456946662,"identity":"8c4bd8cd-779a-4b6e-9b90-9b7b73716455","order_by":7,"name":"Nishant A. Deshmukh","email":"","orcid":"","institution":"Indian Council of Agricultural Research-National Research Centre for Grapes","correspondingAuthor":false,"prefix":"","firstName":"Nishant","middleName":"A.","lastName":"Deshmukh","suffix":""},{"id":456946663,"identity":"754a2876-279d-465b-9426-20258b35ff8d","order_by":8,"name":"Kaushik Banerjee","email":"","orcid":"","institution":"Indian Council of Agricultural Research-National Research Centre for Grapes","correspondingAuthor":false,"prefix":"","firstName":"Kaushik","middleName":"","lastName":"Banerjee","suffix":""}],"badges":[],"createdAt":"2025-05-06 12:08:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6602877/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6602877/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82917996,"identity":"1da7cdac-860b-4caa-bbe8-5fa1caaae82a","added_by":"auto","created_at":"2025-05-16 16:40:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1308609,"visible":true,"origin":"","legend":"\u003cp\u003eFungal and bacterial endophytes isolated, identified, and used for bioformulations development and field evaluations against powdery mildew of grapevine cv. Sarita Seedless at ICAR-National Research Centre for Grapes, Pune experimental farm.\u003c/p\u003e","description":"","filename":"2.Figures1.png","url":"https://assets-eu.researchsquare.com/files/rs-6602877/v1/e1172a04a2c27da519507d9c.png"},{"id":82918275,"identity":"7cf25fd1-f744-427d-840d-8119a80ecad2","added_by":"auto","created_at":"2025-05-16 16:48:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":155050,"visible":true,"origin":"","legend":"\u003cp\u003eThe phylogenetic analysis of the endophytic microbes was inferred using the Neighbor-Joining method. The bootstrap consensus tree inferred from 1000 replicates. The evolutionary distances were computed using the Maximum Composite Likelihood method in MEGA11. (A) Six fungal endophytic fungi and (B) six endophytic bacteria from the present study indicated in bold letters.\u003c/p\u003e","description":"","filename":"2.Figures2.png","url":"https://assets-eu.researchsquare.com/files/rs-6602877/v1/8d5db760c42c30cbc2af5037.png"},{"id":82917997,"identity":"bacf9626-d796-488a-9d88-ba2ffef55e6e","added_by":"auto","created_at":"2025-05-16 16:40:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":74648,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of endophytic \u003cem\u003eTrichoderma\u003c/em\u003e, \u003cem\u003eCytospora,\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003ebioformulations on single bunch weight and yield of grapes/vine during 2022-23 and 2023-24 crop season.\u003c/p\u003e","description":"","filename":"2.Figures4.png","url":"https://assets-eu.researchsquare.com/files/rs-6602877/v1/e9283d69542fda0a8f7db5d2.png"},{"id":82918276,"identity":"67d8d2fa-3fb6-4f2b-b566-8bbd87facdf7","added_by":"auto","created_at":"2025-05-16 16:48:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":63845,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of endophytic \u003cem\u003eTrichoderma\u003c/em\u003e, \u003cem\u003eCytospora\u003c/em\u003e, and \u003cem\u003eBacillus\u003c/em\u003especies on the shelf life of grapes cv. Sarita Seedless during 2022-23 and 2023-24.\u003c/p\u003e","description":"","filename":"2.Figures5.png","url":"https://assets-eu.researchsquare.com/files/rs-6602877/v1/dec101ebed5b99c6843c5f5e.png"},{"id":82918001,"identity":"347e339d-1b6d-4fcf-92e6-88d4ed91c562","added_by":"auto","created_at":"2025-05-16 16:40:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":37669,"visible":true,"origin":"","legend":"\u003cp\u003eBio-efficacy of fungal and bacterial endophytes bioformulations on percent disease index on leaves and berries during the 2022-23 and 2023-24 crop seasons in grapevine cv. Sarita Seedless.\u003c/p\u003e","description":"","filename":"2.Figures6.png","url":"https://assets-eu.researchsquare.com/files/rs-6602877/v1/63d031a78c2e2f69aac678d5.png"},{"id":82918003,"identity":"8652cf5c-906d-4b01-a89c-75d1823f0231","added_by":"auto","created_at":"2025-05-16 16:40:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":419842,"visible":true,"origin":"","legend":"\u003cp\u003eResidue analysis in the final harvest of grapevine cv. Sarita Seedless under various treatments to determine the presence of multiple pesticides and their compliance with maximum residue limits.\u003c/p\u003e","description":"","filename":"2.Figures7.png","url":"https://assets-eu.researchsquare.com/files/rs-6602877/v1/8fc1bb6fd732b38ffd309f4e.png"},{"id":84687999,"identity":"365be2a5-fbd9-4f20-aebe-5bb109097d99","added_by":"auto","created_at":"2025-06-16 09:17:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4529510,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6602877/v1/bb7780e8-1ab9-42b6-a752-0c97ff212032.pdf"},{"id":82917998,"identity":"b0424a48-16f1-494a-bc67-ce1f75491f34","added_by":"auto","created_at":"2025-05-16 16:40:10","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":60829,"visible":true,"origin":"","legend":"","description":"","filename":"4.SupplemetaryTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-6602877/v1/2b27be44dd3cadf7161cc7df.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characterization of grapevine endophytic microbes and field evaluation of bioformulations against powdery mildew disease with residue-compliant grape production ","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eGrapevine (\u003cem\u003eVitis vinifera\u003c/em\u003e L.) is a significant fruit crop grown worldwide. Grapevine is a cultivation aimed at various purposes like fresh consumption, raisins, juice, and wine production. Consumption of grapes benefits human health due to their nutritional and medicinal properties (Garcia Conesa et al. 2023). Worldwide, grapes are grown on around 7.5 M hectares by 79\u0026nbsp;million MT of final produce (FAO, 2023; APEDA, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e-24). Grapes are planted on about 171 thousand ha with a total output of 3781 thousand MT in India (FAO, 2023; APEDA, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e-24). In India, more than 90% area is devoted to table grapes, followed by raisin and wine production. (Sawant et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Kanitkar et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). During 2023\u0026ndash;2024, India exported about 2,63,075.67 metric tons of grapes costing around 305.66\u0026nbsp;million USD to many countries (APEDA, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e-24). Grapevine cultivation worldwide is becoming significant because of the incidence of several biotic constraints under changing climatic conditions, causing huge losses for grapevine industries by reducing quality and financial loss (Bove et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Among the biotic constraints, powdery mildew caused by \u003cem\u003eErysiphe necator\u003c/em\u003e is an economically important disease affecting grapevine production and productivity for domestic and international markets (Gadoury et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Vinothini et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The commercially cultivated grapevine belongs to the genus \u003cem\u003eVitis\u003c/em\u003e and the \u003cem\u003eVinifera\u003c/em\u003e species is widely known for its susceptibility against powdery mildew disease (Fedorina et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Throughout the growing season, powdery mildew affects the leaves, canes, tendrils, inflorescences, rachis, and bunches during dry and cool conditions (Gadoury et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Initially, yellow spots appear on the young leaves, and later white-greyish powdery masses appear on both sides of the older foliage. Diseased young foliage shows curling and inflorescence results in poor fruit set, leading to premature fruit drop. The severely affected berries show cracking and rot (Thind et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Gadoury et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The \u003cem\u003eE. necator\u003c/em\u003e survives in humid conditions, with temperatures ranging from 20-30\u003csup\u003eo\u003c/sup\u003eC, relative humidity of more than 45%, and intermittent rainfall (Gadoury et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eManagement of powdery mildew becomes very essential to avoid crop loss. Therefore, the application of chemicals becomes important during favourable climatic conditions. Constant use of fungicides results in the advancement of resistance in phytopathogens, deposits on the bunches, and their harmful effect on living creatures (Pathak et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). To address these issues, the application of potential microbes that originated as epiphytes, endophytes, and rhizospheric microbes having multiple beneficial effects, especially disease management, and bioremediation of residues, needs to be explored (Holkar et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This approach was found to be environment-friendly and sustainable for grapevine disease management. Grapevine powdery mildew disease has been managed efficiently by beneficial biological control agents (BCAs) \u003cem\u003eviz\u003c/em\u003e., \u003cem\u003eAmpelomyces quisqualis\u003c/em\u003e, \u003cem\u003eTrichoderma asperelloids T. harzianum, T. afroharzianum, Bacillus subtilis\u003c/em\u003e, and \u003cem\u003eBrevibacillus brevis\u003c/em\u003e (Sawant et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Sawant et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sawant et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Caffi et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Avan et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Jena et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Saha et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEndophytic microbes are more diverse bio-resources that benefit host plants as biocontrol agents, provide tolerance to various stresses under changing climatic conditions, and promote plant growth (Jia et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xie et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Joshi et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003eb\u003c/span\u003e; Holkar et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Saha et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kaya et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Grapevine endophytic microbes have been widely used in managing other grapevine diseases which was recently reviewed (Holkar et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Little or no data is presented on applying fungal endophytes for powdery mildew disease management and producing residue-compliant grapes worldwide. Therefore, the present investigation was undertaken to evaluate a diverse group of fungal and bacterial endophytes against powdery mildew management and residue bioremediation. This study will help to create awareness among grape growers to increase the share of BCAs, especially the multiple effects of endophytic microbes for quality and low-input cost production.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eSample\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003eCollection\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental field of ICAR-NRC for Grapes, Pune (8\u0026deg; 29\u0026prime; 570\u0026prime;\u0026prime; N latitude and 73\u0026deg; 59\u0026prime; 168\u0026prime;\u0026prime; E longitude) is at 559 meters altitude from sea level. Different plant parts were collected from grapevine cvs. Manik Chaman (Berries), Dogridge rootstock (stem), Sauvignon Blanc (leaf), Cabernet Sauvignon (leaf, roots, and berries), and wild relative \u003cem\u003eVitis\u003c/em\u003e \u003cem\u003erotundifolia\u003c/em\u003e (leaf). The healthy berry samples (mature and immature) of the grapevine cv. Muscat were collected from the Grape Research Station (GRS), experimental field, Theni, Tamil Nadu state (coordinates: 90 45\u0026rsquo; 26.685\u0026rsquo;\u0026rsquo;N latitude and 770 20\u0026rsquo; 58.737\u0026rsquo;\u0026rsquo;E longitude) at 435.05 meters.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation and Purification of Endophytes\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe collected various plant parts were cleaned using fluent tap water, soaked in a 3% NaoCl (sodium hypochlorite) for three min, and later washed using 70% ethyl alcohol for 30 sec. After rinsing with sterile distilled water (SDW) four times and blotting dry on sterile filter paper (Whatman, Himedia, Germany) for five min. Cleaned leaves and stems were segmented into five mm-sized pieces using a sterilized cork-borer and scissors, respectively. Sterilized samples (leaf and stem) and berries, four samples of each, were inoculated in each Petri dish poured with PDA and NA medium (Himedia, India), approximately 20 ml in each plate, along with 0.2 g/l chloramphenicol. These Petri dishes were then kept for incubation for four days at 26 \u0026deg;C \u003cu\u003e+\u003c/u\u003e 2\u003csup\u003eo\u003c/sup\u003eC for growth. After the incubation period, endophytes obtained from the plates were identified based on their specific growth characteristics on the media (Joshi et al., 2019). The fungal endophytes were purified using the hyphal tip of endophytic fungi by subsequent sub-culturing five times on new Petri dishes poured with PDA medium. Similarly, the ooze out bacteria from leaf cuts were purified on new Petri dishes containing NA medium (Joshi et al. 2019a). Spore morphologies and colony characteristics of \u003cem\u003eTrichoderma\u003c/em\u003e isolates were studied based on phialide cell arrangement. The mother cultures of all isolates were preserved in sterile broth containing 50% glycerol and stored at -80\u0026deg;C for further use. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroscopic Detection of Fungal Endophytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIsolated endophytes were prepared for microscopic examination using a sterile glass slide, and needle, and stained with methyl blue and lactophenol, and observed under a microscope (Leica model: DM750, Microsystems, Germany) with 10X, 40X, and 100X magnifications. For optimal clarity at 100X magnification, the immersion oil technique was used. A microscopic study was conducted as mentioned by Jaklitsch et al. (2012). The purified EB strains were subjected to morphological characterization (Zhao et al. 2016; Urbez-Torres et al. 2020). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphological and Biochemical Characterization of Bacterial Endophytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour EB isolates used in this study were earlier characterized for their morphological, biochemical, and molecular characterization (Holkar et al. 2024). In contrast, the other three\u0026nbsp;EB strains were characterized in the present study for colony characteristics, Gram\u0026rsquo;s reaction (Hucker and Conn 1923), biochemical parameters, and 16S rRNA sequence data. All characteristics of these isolates were studied by referring to Bergey et al. (1939). Each test was carried out twice, with three replications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA Isolation and PCR Amplification of Fungal and Bacterial Endophytes\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExtracted the total genetic material from six fungal endophytes from 100 mg of the 7-day-old culture of endophytes using the protocol described by the DNeasy Plant Mini Kit (Qiagen, Germany). Further, isolated endophyte mat (fungi) was ground to a fine dust using a sterilized mortar-pestle with liquid N\u003csub\u003e2\u003c/sub\u003e. One milliliter of aqueous phase was added to another 1.5 ml microcentrifuge tube and centrifuged at 14,000 rpm for 5 min. The\u0026nbsp;aqueous phase was transferred to a sterilized 1.5 ml microcentrifuge tube, and the subsequent steps were described. DNA elution, the\u0026nbsp;total genetic material was stored in a deep freezer (-20\u0026deg;C). The nuclear ribosomal DNA ITS (internal transcribed spacer) endophytes were amplified using the universal primer pair ITS1 and ITS4 (White et al. 1990).\u003c/p\u003e\n\u003cp\u003ePCR was carried out using a PCR machine (GeneAmp 9700). Total 25 \u0026micro;l reaction recipe composed of 0.3 \u0026micro;l of each primer (100 ng), 0.75 \u0026micro;l of 10 mM dNTPs, 0.25 \u0026micro;l of Taq DNA polymerase (Thermo Fisher Scientific, USA; 5 U/\u0026micro;l), 4.5 \u0026micro;l of DNA template (~2.5 \u0026micro;g), and 2.5 \u0026micro;l of 10\u0026times; buffer. The PCR profile includes 10 min denaturation at 94\u0026deg;C at the initial stage, later 35 rounds of subsequent denaturation for 30 seconds at 94\u0026deg;C, 55 seconds at 55\u0026deg;C annealing, and 1 min at 72\u0026deg;C for extension. A 10-minute incubation at 72\u0026deg;C for final extension. Analyzed the amplicons using 1\u0026times; TAE buffer (90 mM Tris-acetate, 2 mM EDTA, pH 8.0), 1% agarose gel electrophoresis stained with 0.01% ethidium bromide (0.5 \u0026micro;g/ml). Cleaned the amplicons using PCR Purification Kit (QIAquick, Qiagen, Germany) according to the protocol. Amplicons from six endophytes were sent for the double-pass Sanger method at GenOmBio Technologies Pvt. Ltd. Pune, Maharashtra, India.\u003c/p\u003e\n\u003cp\u003eTotal DNA was isolated from of 24 h old grown seven endophytic bacterial strains with the help of the producer\u0026apos;s protocol (Himedia, India). Eluted total DNA was stored in a deep freezer (-20\u0026deg;C). \u0026nbsp;The total DNA was amplified by PCR using the 27F and 1492R universal primer pair corresponding to the 16S rDNA gene (Lane, 1991). The same PCR machine was used for bacterial endophytes with the same PCR reaction recipe, except DNA template. The PCR profile for amplification of 16S rDNA was like ITS primers except annealing temperature at 57 \u0026deg;C for 30 s, and an extension at 72 \u0026deg;C for 90 s. \u0026nbsp;Amplicons were cleaned up and submitted for double-pass Sanger sequencing. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogeny\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong the 13 isolates, 12 isolates showed desired amplification and were subjected to sequencing from GeneOmbio, Technologies Pvt. Ltd., Pune, Maharashtra State, India. Nucleotide sequences were assembled in the SeqMan program (Swindell and Plasterer, 1997). The analyzed sequences were submitted to the NCBI GenBank. The BLASTn analyses (https://blast.ncbi.nlm.nih.gov/) were carried out for sequence homology of six EB strains and six FEs. The sequence identity matrix and alignment were carried out using the ClustalW program using BioEdit software version 1.7 (www.bioedit.software.informer.com). The evolutionary history was inferred using MEGA11 software (Kumar et al., 2016; Tamura et al., 2021) by the Neighbour-Joining method (Saitou and Nei, 1987). A 1000 bootstrap test was undertaken to determine replication percentage (Felsenstein, 1985). The sequences from NCBI GenBank were used for comparative analyses as presented in the phylogenetic tree. \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDevelopment of Bioformulations\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the \u003cem\u003ein vitro\u003c/em\u003e antagonistic effect of these endophytic microbes on the mycelium growth of \u003cem\u003eColletotrichum\u003c/em\u003e \u003cem\u003egloeosporioides\u003c/em\u003e, a causal pathogen of anthracnose in grapevine. Therefore, formulations of six FEs and seven BEs were developed and evaluated for the \u003cem\u003eErysiphe necator\u003c/em\u003e under field conditions, considering direct antagonism of the target pathogen. For the development of the bioformulation of FEs, flattened rice was used as a carrier and applied under field conditions to test their efficacy against powdery mildew disease on grapevine cv. \u003cem\u003eSarita Seedless\u003c/em\u003e during the 2022-23 and 2023-24 crop growing seasons. Briefly, the procedure of preparation of \u003cem\u003eTrichoderma\u003c/em\u003e formulation is as follows. The MnSO₄ (0.4 g), D-(+)-Glucose anhydrous (20 gm), CaCl₂ (0.4 gm), and MgSO₄ (0.4 gm) were dissolved in 1 L of distilled water. Flattened rice 1 Kg was soaked in double-distilled water, and the chemical solution prepared using the chemicals described above was mixed thoroughly. The mixture was sterilized at 121\u0026deg;C under 15 psi for a duration of 40 min in a vertical autoclave machine. After sterilization, the flattened rice was allowed to cool and transferred to sterile aluminum trays of approximately 30 cm x 20 cm size to ensure uniform drying and inoculation. A week-old pure cultures of \u003cem\u003eTrichoderma\u003c/em\u003e species were inoculated onto the flattened rice, and the trays were kept for incubation at 28\u0026ndash;30\u0026deg;C with an absolute moisture of 80\u0026ndash;85% for two weeks. Sufficient moisture, 60% - 70%, was maintained in the inoculated trays by spraying double-distilled water. After two weeks of incubation, \u003cem\u003eTrichoderma\u003c/em\u003e colonized the flattened rice, and mycelial and sporulation growth were observed. These trays were kept for sun drying for 2-3 h. Colonized dried substrate ground to fine dust particles, with the help of a homogenizer, and stained homogeneous fine powder having 1\u0026times;10\u003csup\u003e9\u003c/sup\u003e spores/g. It was used for spraying under field conditions for powdery mildew management. The reproducibility of each formulation was recorded. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSimilarly, for the bacterial formulations, single bacterial colonies were inoculated in a nutrient broth medium for mass production from the bacterial colonies grown on an NA medium. This was incubated at 28\u003cu\u003e+\u003c/u\u003e2 \u003csup\u003eo\u003c/sup\u003eC for 48 h. The liquid formulations with 0.5 OD were used for the field sprays. The spore count of the liquid formulation was maintained at 1 \u0026times; 10\u003csup\u003e11\u003c/sup\u003e CFU/ml. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eField Evaluation of Fungal and Bacterial Bioformulations \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present investigation was conducted at the institute\u0026apos;s experimental farm during the 2022-23 and 2023-24 crop seasons to evaluate the efficacy of isolated endophytes for powdery mildew disease in grape cv. Sarita seedless. Bioformulation evaluation was based on the natural occurrence of the disease. The climatic conditions during both seasons were conducive to the natural occurrence of the disease. The experimental cultivar was eight years old with black soil (Vertisol) having pH of 7.5-7.6. The sprays were undertaken when the first symptoms of the disease were observed on vines. The sprays were scheduled weekly; the vines were treated with sulphur 80% WDG @ 2 g/L as a positive control. For each treatment, three vines were used with three replications by applying a Randomized Block Design (RBD). A knapsack sprayer was used for uniform coverage of endophytic bioformulations on leaves and berries. The percent disease index was observed before scheduling the first spray till one week after the last spray by applying a 0\u0026ndash;4 rating score (Horsfall and Heuberger 1942). A total of 100 random leaves per replication (10 canes and 10 leaves per cane) were observed for the disease and assigned a 0-4 score based on the symptoms and severity. The percent disease index (PDI) was calculated using the score data (Wheeler, 1969). The disease ratings had also been recorded on the bunches using a similar scale used for foliar symptoms. A total of 20 bunches were observed per replication. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Sum of all numerical ratings\u003c/p\u003e\n\u003cp\u003ePercent Disease Index = \u0026nbsp;--------------------------------------------------------------------- x 100\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Number of leaves/bunches observed x maximum grade\u003c/p\u003e\n\u003cp\u003eThe percent disease control (PDC) is calculated as under: \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;PDI in untreated control \u0026ndash; PDI in treatment\u003c/p\u003e\n\u003cp\u003ePDC = ------------------------------------------- x 100\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;PDI in untreated control\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysical and Biochemical Characterization\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe harvested grape bunches of the grapevine cv. Sarita Seedless was subjected to the estimation of physical and biochemical characteristics carried out during the 2022-23 and 2023-24 crop seasons. The physical characteristics, like 50-berry weight, bunch weight, skin thickness, rachis length, number of berries per bunch, berry length, berry diameter, total soluble solids (TSS), acidity, pH, pedicel length, physiological weight loss (PWL), and yield were estimated (Satisha and Somkuwar 2019; Thosar et al. 2020). The biochemical analysis included the evaluation of phenol, tannin, reducing sugars, protein, proline, carbohydrates, and antioxidants (Somkuwar et al. 2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysiological Weight Loss (PWL)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShelf-life analysis of a grapevine sample harvested from cv. Sarita seedless was calculated based on a weight-loss basis. Weight loss was calculated by recording the initial and final weight by applying a formula:\u003c/p\u003e\n\u003cp\u003eWeight loss = [(RI-RII) \u0026divide; RI] x 100\u003c/p\u003e\n\u003cp\u003ewhere, RI= Initial Weight and RII\u0026ndash; Final Weight \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemical Reagents and Preparation of Stock Solution for\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePesticide Residue Extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe certified reference material (CRM) of selected pesticides (98.20-99.50% purity) was used (Augsburg, Germany). Analytical quality ethyl acetate (99.8%), acetonitrile (98.4%), LC-MS grade methanol, iso-octane (99.5%), acetic acid (99%), Carbon disulfide (CS2, 99.9% purity), and formic acid (95%), hydrochloric acid (35%), diethylene glycol (DEG), Sodium sulphate (Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e), ammonium formate (Merck India Ltd., Mumbai), and primary secondary amine (PSA) (Agilent Technologies, Santa Clara, CA, USA). The individual stock solutions of certified pesticide standards (10 \u0026plusmn; 0.10 mg) were prepared in methanol (10 \u0026plusmn; 0.10 g) and stored at -20\u0026deg;C for fungicides, insecticides, and plant growth regulators. Thereafter, intermediate (10 \u0026micro;g/mL) and working (1 \u0026micro;g/mL) standard solutions for matrix-matched and solvent standard calibrations were made through serial dilution in methanol.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCS₂ solution (stock) was prepared for \u003cstrong\u003ecarbon disulfide for di-thiocarbamates\u003c/strong\u003e by pipetting 10 \u0026micro;L of CS₂ standard into 10 mL of iso-octane. Then, working solutions (1 and 0.1 mg/kg) were obtained by the serial dilution method with iso-octane solvent. A separate thiram stock solution was prepared by mixing 10 \u0026micro;L of the standard in C\u003csub\u003e4\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e(ethyl acetate). After that, a 1 mg/kg working solution of thiram was then prepared by serial dilution. The reaction mixture \u003cstrong\u003efor dithiocarbamates\u003c/strong\u003e was prepared by dissolving 30 g of stannous (II) chloride in 1000 mL of 35% HCL, followed by the gradual addition of 1000 mL of water and sonication for 2 minutes\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample Preparation and Analytical Instruments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the detection of residues of fungicide and insecticide, a homogenized grape sample (10.00 \u0026plusmn; 0.10 g) was weighed into a centrifuge tube (50 mL). The 10 mL of\u0026nbsp;C\u003csub\u003e4\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was then added to the homogenized grape sample, and thoroughly mixed at 15,000 rpm for 1 minute. Anhydrous sodium sulfate (10 g) was added next, and the homogenization was repeated under the same conditions. The sample extract was centrifuged at 5000 rpm for 5 min and subsequently processed by LC-MS/MS (Shimadzu UFLC XR system coupled with mass spectrometer (AB Sciex API 4000 QqQLIT) and GC-MS/MS (Shimadzu TQ8040 GC-MS/MS system (Shimadzu Corporation, Japan) armed with an Rxi\u0026reg;-5Sil MS column (Restek Corporation, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSimilarly, for detecting residues of ethephon, the homogenized sample (10.00 \u0026plusmn; 0.10 g) was accurately weighed, and 20 mL of acidified methanol (1% formic acid) was added. Further, the mixture was mixed for 2 min and centrifuged for 5 min at 5000 rpm. Then, 500 \u0026micro;L of the separated aqueous layer was combined with 500 \u0026micro;L of acetonitrile for analysis of ethephon residues. Extraction of the di-thiocarbamate residue analysis, the grape berries (\u0026plusmn;25.01 g) were placed in a glass bottle with having 250 ml capacity. After that,\u0026nbsp;a 75 mL recipe (30 g Stannous (II) chloride in 1000 mL HCL + 1000 mL water) was added, followed by 25 mL of iso-octane, and the bottle was capped quickly. After that, the bottle was incubated in a water bath at 80\u0026deg;C and shaken intermittently every 20 minutes for 1 hr. Then the bottle was dipped in ice-cold water to cool it down to \u0026lt;20 \u0026deg;C. 1 mL of the upper iso-octane layer aliquot was added to a new tube and subjected to centrifugation for 5 min at 10 \u0026deg;C. After injecting the aliquot into the GC, di-thiocarbamate residues were calculated as CS\u003csub\u003e2\u003c/sub\u003e using GC/MS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOptimized parameters for individual pesticides, both for GC-MS/MS and LC-MS/MS, are provided (Supplementary Table 1). In both the ionization modes (positive and negative), mass spectrometric identification was carried out. Information procurement and estimation for LC-MS/MS were performed using Analyst software (v1.7.1). However, for di-thiocarbamates, mass spectrometry was operated in SIM mode with a temperature of 40-290\u0026deg;C (Supplementary Table 2). Argon was used as the collision gas, and mass acquisition began at 4 minutes. Data acquisition and processing GC-MS/MS was performed using LabSolutions\u0026reg; software (Version 4.50, Japan). The analytical method SANTE/ 11312/2021\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewas followed (SANTE, 2021).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe field observations on treatment were repeated three times and recorded. The recorded data was analyzed for the calculation of analysis of variance (ANOVA) by randomized block design with the help of OPSTAT, an online tool. The initial recorded data was converted to arcsine values and used for ANOVA. The standard error of mean, standard error of deviations, and critical difference (C.D.) values were estimated. \u0026nbsp;The level of significance is indicated at the bottom of the tables.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMorphological Identification of Fungal and Bacterial Endophytes\u003c/h2\u003e \u003cp\u003eThe 13 endophytic isolates were successfully purified, which originated from berries, stems, roots, and leaves of the grapevine cv. Manik Chaman, dogridge, Muscut, Sauvignon Blanc, and \u003cem\u003eVitis rotundifolia\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplementary Table\u0026nbsp;3). Based on the morphological key characteristics, fungal endophytes \u003cem\u003eviz\u003c/em\u003e., CSBY-2, CSBY-4, CSBY-8, MCBY-2, and DRRS-1 were identified as \u003cem\u003eTrichoderma\u003c/em\u003e sp. All the fungal endophytes produced distinctive green to olive-green colonies, powdery texture, and rapid growth on the PDA medium. Based on the microscopic observations endophytic \u003cem\u003eTrichoderma\u003c/em\u003e isolates showed septate hyphae, branched conidiophores, and globose conidia.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe four EB strains \u003cem\u003eviz\u003c/em\u003e., SB5, SB2, CS2, and RF1 were earlier characterized (Holkar et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), whereas three EB strains \u003cem\u003eviz\u003c/em\u003e., RT7, RB1, and GB2 were characterized in the present study. The EB strains were Gram\u0026rsquo;s Gram-positive, mucoid colonies, with convex elevations having circular to irregular shapes and white-colored colonies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMolecular Identification of Endophytic Microbes\u003c/h2\u003e \u003cp\u003eBased on ITS sequence information, the endophytic \u003cem\u003eTrichoderma\u003c/em\u003e isolates \u003cem\u003eviz\u003c/em\u003e., CSBY2, CSBY8, and MCBY2 were identified as \u003cem\u003eTrichoderma asperellum.\u003c/em\u003e In contrast, CSBY4, DRRS1, and MCBY1 were identified as \u003cem\u003eT. asperelloides\u003c/em\u003e, strains \u003cem\u003eviz\u003c/em\u003e., Sauvignon Blanc \u0026minus;\u0026thinsp;5, -2, Crimson Seedless \u0026minus;\u0026thinsp;2, and Rotundifolia \u0026minus;\u0026thinsp;1 were recognized as \u003cem\u003eBacillus subtilis\u003c/em\u003e, whereas RT7, RB1, and GB2 strains were recognized as \u003cem\u003eB. mojavensis\u003c/em\u003e, and \u003cem\u003eB. licheniformis\u003c/em\u003e, respectively (Supplementary Table\u0026nbsp;3 and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Sequence information of all the isolates was deposited in the GenBank and received the accession numbers for each isolate.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of Endophytic\u003c/b\u003e \u003cb\u003eTrichoderma\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eBacillus\u003c/b\u003e \u003cb\u003eSpecies on Physical Parameters of Grapes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe efficacy of various \u003cem\u003eTrichoderma\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e-based bio-formulations in improving the physical parameters of cv. Sarita Seedless was evaluated during the 2022-23 and 2023-24 crop seasons. T1 to T16 treatments with untreated control (UTC) were followed. During the 2022\u0026ndash;2023 crop growing season, treatments showed positive results in enhancing specific physical traits. Results revealed that T4 (\u003cem\u003eBacillus licheniformis\u003c/em\u003e strain RB1) treatment improved average bunch weight (450.4 g), pH (3.27), and yield (13.962 kg/vine); T6 (\u003cem\u003eBacillus mojavensis\u003c/em\u003e strain RT7) increased 50-berry weight (224.8 g), berry length (2.75 mm), and berry diameter (1.56 mm); T10 (\u003cem\u003eCytospora\u003c/em\u003e species MCBY1 isolate) increased the number of berries per bunch (95); T15 (\u003cem\u003eBacillus subtilis\u003c/em\u003e strain SB2\u0026thinsp;+\u0026thinsp;\u003cem\u003eCytospora\u003c/em\u003e species MCBY1 isolate) enhanced skin thickness (0.27 mm) and rachis length (238.88 mm); T8 (\u003cem\u003eTrichoderma viride\u003c/em\u003e isolate DRRS1 ) improved pedicel length (13.54 mm) and single bunch weight (152.26 g); T1 (\u003cem\u003eBacillus\u003c/em\u003e spp. strain GB2) increased TSS (21.4), and T13 (\u003cem\u003eT. viride\u003c/em\u003e isolate DRRS1\u0026thinsp;+\u0026thinsp;\u003cem\u003eB. subtilis\u003c/em\u003e strain SB2) reduced acidity (0.87) as described in (Supplementary Table\u0026nbsp;4 and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). During the 2023\u0026ndash;2024 crop-growing season, different treatments showed improved physical parameters. T5 (\u003cem\u003eB. subtilis\u003c/em\u003e strain SB2) increased average bunch weight (556.67 g) and TSS (23.35); T3 (\u003cem\u003eT. asperellum\u003c/em\u003e isolate MCBY2) enhanced 50-berry weight (249 g) and pedicel length (14.71 mm); T12 (\u003cem\u003eT. asperellum\u003c/em\u003e isolate MCBY2\u0026thinsp;+\u0026thinsp;\u003cem\u003eB. subtilis\u003c/em\u003e strain SB2) improved the number of berries per bunch (114); T7 (\u003cem\u003eT. asperelloides\u003c/em\u003e isolate CSBY4\u0026thinsp;+\u0026thinsp;\u003cem\u003eB. subtilis\u003c/em\u003e strain SB5) increased skin thickness (0.38 mm); T15 (\u003cem\u003eT. asperelloides\u003c/em\u003e isolate CSBY4\u0026thinsp;+\u0026thinsp;\u003cem\u003eT. viride\u003c/em\u003e isolate DRRS1\u0026thinsp;+\u0026thinsp;\u003cem\u003eT. asperellum\u003c/em\u003e isolate MCBY2\u0026thinsp;+\u0026thinsp;\u003cem\u003eB. subtilis\u003c/em\u003e, SB5\u0026thinsp;+\u0026thinsp;\u003cem\u003eB. subtilis\u003c/em\u003e, SB2\u0026thinsp;+\u0026thinsp;\u003cem\u003eB. subtilis\u003c/em\u003e strain CS2) lengthened rachis length (249.78 mm) and raised acidity (2.6); T16 (positive control with sulphur) improved single bunch weight (184.89 g); T6 increased berry length (2.7 mm); T9 (\u003cem\u003eT. asperellum\u003c/em\u003e isolate MCBY2\u0026thinsp;+\u0026thinsp;\u003cem\u003eB. subtilis\u003c/em\u003e strain SB5) enhanced berry diameter (1.79 mm) and pH (3.96); and T1 (\u003cem\u003eT. asperelloides\u003c/em\u003e isolate CSBY4) increased yield (14.377 kg/vine), as detailed in (Supplementary Table\u0026nbsp;5 and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). During both the crop growing season, bioformulations of RB1, CSBY4, MCBY2, SB5 and DRRS1 showed promising results in all the physical parameters.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eEffects of Endophytic Bioformulations on Biochemical Parameters of Grapes\u003c/h2\u003e \u003cp\u003eBiochemical observations from grape samples of cv. Sarita Seedless, collected during two consecutive crop growing seasons \u003cem\u003ei.e.\u003c/em\u003e, 2022\u0026ndash;2023 and 2023\u0026ndash;2024 at ICAR-NRCG, Pune experimental farm. Multiple biochemical traits including, phenol, tannin, reducing sugar, protein, proline, carbohydrate, and antioxidants across 16 different treatments (T1\u0026ndash;T16) and untreated control (UTC) were compared. The data indicated that phenol (mg/g) content ranged from 0.45 (T1: GB2) to 0.63 (T10: MCBY1) and 0.14 (T15: CSBY4\u0026thinsp;+\u0026thinsp;DRRS1\u0026thinsp;+\u0026thinsp;MCBY2\u0026thinsp;+\u0026thinsp;SB5\u0026thinsp;+\u0026thinsp;SB2\u0026thinsp;+\u0026thinsp;CS2) to 0.86 (T2: DRRS1) during the years 2022-23 and 2023-24, respectively (Supplementary Table\u0026nbsp;6). Tannin levels ranged from 0.57 (T1) to 0.82 (T10) and 0.51 (T13) to 0.89 (T10) during the same year. Reducing sugar content was recorded higher in 2022-23 ranging from 19.03 (T4: RB1) to 59.1 (T3: SB2). It was recorded lowest during the 2023-24 crop season, ranging from 14.87 (T4: SB5) to 43.75 (T9: MCBY2\u0026thinsp;+\u0026thinsp;SB5). In the year, 2022-23 protein level ranged from 1.26 (T16: PC with Sulphur) to 1.84 (T2: SB5), and in 2023-24 it was 0.74 (UTC) to 1.97 (T2: DRRS1). Proline levels varied significantly, ranging from 1.36 (T10: MCBY1) to 5.59 (T5: RF1) and 3.45 (UTC) to 5.14 (T12: MCBY2\u0026thinsp;+\u0026thinsp;SB2) during 2022-23 and 2023-24, respectively (Supplementary Table\u0026nbsp;6).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of Endophytic\u003c/b\u003e \u003cb\u003eTrichoderma\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eBacillus\u003c/b\u003e \u003cb\u003eIsolates on Shelf-Life of Grapes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBenefits of bioformulations on the lifespan of grape berries were carried out based on loss in initial weight during five days at room temperature, 28\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;2 \u003csup\u003eo\u003c/sup\u003eC, as indicated in Supplementary Table\u0026nbsp;7. It was noted that T6 (RT7) had the lowest weight loss in 2022\u0026ndash;2023 at 2.17%, but this increased significantly to 5.31% in 2023\u0026ndash;2024, revealing one of the better-performing treatments in improving the shelf-life of grape berries. T5 (RF1) also demonstrated good performance, with 3.66% weight loss in 2022\u0026ndash;2023, to 4.93% (T5: SB2) in 2023\u0026ndash;2024, indicating a relatively stable shelf-life across both years. On the other hand, T15 exhibited the highest weight loss in 2023\u0026ndash;2024 at 8.43%, compared to 4.30% in 2022\u0026ndash;2023. The UTC also showed a significant increase in weight loss from 5.75\u0026ndash;6.17% in 2022-23 and 2023-24. The treatments like T4, T7, T8, and T10 showed a moderate increase in weight loss between the two seasons. For instance, T4 had 5.09% weight loss in 2022\u0026ndash;2023, which increased to 5.69% in 2023\u0026ndash;2024 (Supplementary Table\u0026nbsp;7). The treatment with DRRS1\u0026thinsp;+\u0026thinsp;SB2 provided the minimum weight loss (4.42%) in 2022-23 whereas, SB2 alone in 2023-24 showed 4.93% weight loss, therefore the application of SB2 was found effective in improving the shelf-life with minimum weight loss.\u003c/p\u003e \u003cp\u003eThe standard error ranged from 51.99 to 79.564 for initial weight and from 51.192 to 72.542 for final weight, with coefficients of variation (CV%) between 22.065% and 26.645%. Statistical significance for weight loss was observed at a 5% level of significance (p\u0026thinsp;=\u0026thinsp;0.00542 for 2022\u0026ndash;2023), highlighting the effectiveness of specific treatments, particularly T6, in reducing weight loss and enhancing the post-harvest shelf-life of grapes (Supplementary Table\u0026nbsp;7 and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eBio-Efficacy of Endophytes against Powdery Mildew Disease of Grapes\u003c/h2\u003e \u003cp\u003eDuring both the crop growing seasons 2022-23 and 2023-24, a total of 13 bioformulations were field evaluated against powdery mildew. Based on the in vivo evaluations during 2022-23, only three fungal and three bacterial bio-formulations and their combinations were evaluated during 2023-24. The per cent disease index (PDI) during 2022-23 ranged from 25.18\u0026ndash;68.83% on leaves and 23.82\u0026ndash;67.19% on bunches (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Likewise, during 2023-24, PDI ranged from 27.99\u0026ndash;66.83% on leaves and 21.46\u0026ndash;66.59% on bunches (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). All 13 bioformulations showed per cent disease control (PDC) as compared to UTC. The PDC during the 2022-23 season ranged from 12.49\u0026ndash;63.41% and 44.5\u0026ndash;64.56% on leaves and bunches, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Likewise, the PDC during the 2023-24 season ranged from 26.0\u0026ndash;63.18% and 44.97\u0026ndash;67.7% on leaves and bunches, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). During 2022-23 crop season, five bioformulations viz., T1 (GB2), T3 (SB2), T5 (RF1), T6 (RT7) and T15 (SB2\u0026thinsp;+\u0026thinsp;MCBY1) showed maximum PDC ranged from 42.55\u0026ndash;63.41% and 49.73\u0026ndash;64.56% on leaves and berries, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Similarly, during 2023-24 crop season, five treatments \u003cem\u003eviz\u003c/em\u003e., T3 (MCBY2), T5 (SB2), T13 (CSBY4\u0026thinsp;+\u0026thinsp;DRRS1\u0026thinsp;+\u0026thinsp;MCBY2), T14 (SB2\u0026thinsp;+\u0026thinsp;SB5\u0026thinsp;+\u0026thinsp;CS2) and T15 (CSBY4\u0026thinsp;+\u0026thinsp;DRRS1\u0026thinsp;+\u0026thinsp;MCBY2\u0026thinsp;+\u0026thinsp;SB2\u0026thinsp;+\u0026thinsp;SB5\u0026thinsp;+\u0026thinsp;CS2) showed to be potential treatments in PDC ranged from 58.11\u0026ndash;63.18% and 48.60\u0026ndash;67.7% on leaves and bunches, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). During both the crop growing seasons, MCBY2 (60.93% and 67.7%), SB2 (61.57% and 64.56%) isolates performed to be the best among all the bioformulations field evaluated. Moreover, RF1 (63.41%), GB2 (61.04%), and combinations of \u003cem\u003eT. viride\u003c/em\u003e isolate DRRS1, \u003cem\u003eT. asperellum\u003c/em\u003e isolate MCBY2 and \u003cem\u003eT. asperelloides\u003c/em\u003e isolate CSBY4 (63.18%) showed promising results in managing the disease on leaves (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). All the formulations were tested for their compatibility with the chemical fungicides, it was found to be highly compatible with propineb 70 WP, and a combination of 5% kasugamycin and 45% WP copper oxychloride, and 18% kresoxim methyl with 54% mancozeb. Whereas, the formulations were moderately compatible with copper oxychloride 50 WP, 8.3% azoxystrobin and 66.7% WG mancozeb, and 47.15% copper sulphate with 30% mancozeb. These formulations were not compatible with the chemical fungicides, \u003cem\u003eviz\u003c/em\u003e., fluopyram 200\u0026thinsp;+\u0026thinsp;tebuconazole 200 SC, carbendazim 50 WP, thiophanate methyl 70 WP, carbendazim 12% + mancozeb 63% WP. The solid-state formulations were stable for six months with a spore count of 10\u003csup\u003e11\u003c/sup\u003e/ml, after six months spore count was reduced to 10\u003csup\u003e10\u003c/sup\u003e/ ml under refrigerated conditions. \u003cem\u003eTrichoderma\u003c/em\u003e solid-state formulations retained their viability for 12 months with a drop in one log. Whereas, the bacterial liquid state formulations were found to be viable for six months when stored under refrigerated conditions.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eField evaluation of \u003cem\u003eTrichoderma\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e formulations during the 2022-23 crop growing season in grapevine cv. Sarita Seedless at experimental farm of ICAR-NRCG, Pune, Maharashtra, India.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003ePDI on leaves (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePDC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c12\" namest=\"c8\"\u003e \u003cp\u003ePDI on Bunches (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003ePDC\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBefore spray\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAfter 1st spray\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAfter 2nd spray\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAfter 3rd spray\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAfter 4th spray\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eBefore spray\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAfter 1st spray\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eAfter 2nd spray\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eAfter 3rd spray\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eAfter 4th spray\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.76\u003c/p\u003e \u003cp\u003e(13.83)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.21\u003c/p\u003e \u003cp\u003e(18.41)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.04\u003c/p\u003e \u003cp\u003e(22.71)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.11\u003c/p\u003e \u003cp\u003e(26.06)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e26.81\u003c/p\u003e \u003cp\u003e(33.61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e61.04%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.91\u003c/p\u003e \u003cp\u003e(9.18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e11.45\u003c/p\u003e \u003cp\u003e(21.17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e20.44\u003c/p\u003e \u003cp\u003e(26.11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e26.17\u003c/p\u003e \u003cp\u003e(30.21)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e30.16\u003c/p\u003e \u003cp\u003e(36.16)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e55.12%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.01\u003c/p\u003e \u003cp\u003e(14.31)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.96\u003c/p\u003e \u003cp\u003e(18.74)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.48\u003c/p\u003e \u003cp\u003e(25.81)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24.00\u003c/p\u003e \u003cp\u003e(31.18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e27.76\u003c/p\u003e \u003cp\u003e(33.21)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e59.66%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.22 (11.95)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e13.32 (21.56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e20.68 (24.31)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e24.42 (28.16)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e29.44\u003c/p\u003e \u003cp\u003e(33.98)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e56.18%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.77\u003c/p\u003e \u003cp\u003e(14.94)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.32\u003c/p\u003e \u003cp\u003e(17.24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.21\u003c/p\u003e \u003cp\u003e(23.86)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24.60\u003c/p\u003e \u003cp\u003e(31.18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e26.44\u003c/p\u003e \u003cp\u003e(33.36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e61.57%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.08 (11.23)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e14.49 (21.19)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e16.20 (26.31)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e20.16 (34.32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e23.82\u003c/p\u003e \u003cp\u003e(38.69)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e64.56%\u003c/p\u003e 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align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.31\u003c/p\u003e \u003cp\u003e(24.72)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22.13\u003c/p\u003e \u003cp\u003e(30.66)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25.18\u003c/p\u003e \u003cp\u003e(34.41)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e63.41%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.50 (12.18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e16.76 (23.09)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e22.11 (28.60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e31.17 (33.88)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e33.78\u003c/p\u003e \u003cp\u003e(37.96)\u003c/p\u003e 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colname=\"c5\"\u003e \u003cp\u003e26.81\u003c/p\u003e \u003cp\u003e(30.71)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e33.07\u003c/p\u003e \u003cp\u003e(37.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e51.96%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.04 (11.96)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e13.71 (21.13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e21.10 (26.51)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e26.55 (30.29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e30.71\u003c/p\u003e \u003cp\u003e(34.81)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e54.30%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.04 (19.46)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.68 (28.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28.64\u003c/p\u003e \u003cp\u003e(32.03)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e37.46 (38.82)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e39.56\u003c/p\u003e \u003cp\u003e(41.12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e42.68%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.90 (11.29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e11.77 (19.14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e24.70 (28.24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e28.16 (35.87)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e33.22\u003c/p\u003e \u003cp\u003e(39.04)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e50.56%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT15\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.04 (16.13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.60 (23.24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.15 (26.71)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26.97 (31.23)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30.86\u003c/p\u003e \u003cp\u003e(38.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e55.13%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.06 (10.07)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e13.15 (21.21)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e19.17 (25.89)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e23.94 (29.84)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e27.41\u003c/p\u003e \u003cp\u003e(32.63)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e59.2%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.80 (20.17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.16\u003c/p\u003e \u003cp\u003e(23.14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.19 (27.37)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26.19 (30.18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e32.48\u003c/p\u003e \u003cp\u003e(33.61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e52.80%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.35 (10.52)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e12.26 (20.41)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e17.12 (25.92)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e27.03 (30.58)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e37.26\u003c/p\u003e \u003cp\u003e(40.40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e44.5%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eUTC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.65\u003c/p\u003e \u003cp\u003e(19.14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.13\u003c/p\u003e \u003cp\u003e(31.76)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.12\u003c/p\u003e \u003cp\u003e(38.55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e66.35\u003c/p\u003e \u003cp\u003e(54.61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e68.83\u003c/p\u003e \u003cp\u003e(58.66)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.51 (12.13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e25.22 (30.95)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e36.23 (37.31)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e53.10 (47.16)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e67.19\u003c/p\u003e \u003cp\u003e(69.98)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSE \u0026plusmn;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e1.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCD 5%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e5.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e5.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCV (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e10.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e10.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e10.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e10.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"13\"\u003e\u003cem\u003eLevel of significance at p-value\u003c/em\u003e\u0026thinsp;\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003e\u0026le;\u003c/span\u003e\u0026thinsp;\u003cem\u003e0.05 except before the spray\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eField evaluation of \u003cem\u003eTrichoderma\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e formulations during the 2023-24 crop growing season (pooled mean of two seasons of respective treatments) in grapevine cv. Sarita Seedless at experimental farm of ICAR-NRCG, Pune, Maharashtra, India\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003ePDI on leaves (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePDC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c12\" namest=\"c8\"\u003e \u003cp\u003ePDI on Bunches (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003ePDC\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBefore spray\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAfter 1st spray\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAfter 2nd spray\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAfter 3rd spray\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAfter 4th spray\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eBefore spray\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAfter 1st spray\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eAfter 2nd spray\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eAfter 3rd spray\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eAfter 4th spray\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.84\u003c/p\u003e \u003cp\u003e(14.93)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.2\u003c/p\u003e \u003cp\u003e(19.59)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.23\u003c/p\u003e \u003cp\u003e(24.66)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23.61\u003c/p\u003e \u003cp\u003e(28.39)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e28.11\u003c/p\u003e \u003cp\u003e(33.61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e57.92%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.04\u003c/p\u003e \u003cp\u003e(9.48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e12.89\u003c/p\u003e \u003cp\u003e(20.27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e21.19\u003c/p\u003e \u003cp\u003e(27.31)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e26.17\u003c/p\u003e \u003cp\u003e(30.21)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e31.46\u003c/p\u003e \u003cp\u003e(36.18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e52.07%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.06\u003c/p\u003e \u003cp\u003e(14.32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.56\u003c/p\u003e \u003cp\u003e(19.22)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.12\u003c/p\u003e \u003cp\u003e(26.45)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24.32\u003c/p\u003e \u003cp\u003e(31.22)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e28.68\u003c/p\u003e \u003cp\u003e(34.18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e57.09%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.21 (11.75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e13.92 (21.82)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e19.73 (26.31)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e24.72 (29.76)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e29.36\u003c/p\u003e \u003cp\u003e(33.91)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e55.87%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.81\u003c/p\u003e \u003cp\u003e(15.03)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.62\u003c/p\u003e \u003cp\u003e(16.98)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.51\u003c/p\u003e \u003cp\u003e(24.91)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22.16\u003c/p\u003e \u003cp\u003e(29.48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e26.09\u003c/p\u003e \u003cp\u003e(33.16)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e60.93%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.17 (10.23)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e13.49 (21.49)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e14.20 (27.30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e17.16 (31.32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e21.46\u003c/p\u003e \u003cp\u003e(38.69)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e67.7%\u003c/p\u003e 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\u003cp\u003e10.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e14.48 (22.33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e21.92 (27.82)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e28.76 (32.30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e35.44\u003c/p\u003e \u003cp\u003e(37.26)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e46.07%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.35\u003c/p\u003e \u003cp\u003e(14.48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.24\u003c/p\u003e \u003cp\u003e(18.92)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.54\u003c/p\u003e \u003cp\u003e(26.56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24.87\u003c/p\u003e \u003cp\u003e(32.43)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e27.99\u003c/p\u003e \u003cp\u003e(37.14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e58.11%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.70 (11.08)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e14.96 (22.71)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e23.17 (28.70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e31.17 (33.88)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e34.71\u003c/p\u003e \u003cp\u003e(38.07)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e44.97%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.93\u003c/p\u003e \u003cp\u003e(16.33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.7\u003c/p\u003e \u003cp\u003e(25.63)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.84\u003c/p\u003e \u003cp\u003e(31.66)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36.54\u003c/p\u003e \u003cp\u003e(36.98)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e42.76\u003c/p\u003e \u003cp\u003e(43.39)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e36.03%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5.80 (13.90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e12.38 (20.52)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e19.20 (25.94)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e23.20 (28.73)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e27.33\u003c/p\u003e \u003cp\u003e(32.83)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e63.00%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.77\u003c/p\u003e \u003cp\u003e(13.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.86\u003c/p\u003e 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colname=\"c7\"\u003e \u003cp\u003e26.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.64 (10.89)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e13.61 (21.58)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e25.16 (30.04)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e32.01 (34.42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e36.20\u003c/p\u003e \u003cp\u003e(39.66)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e45.67%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.62 (14.81)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e 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\u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.97 (11.39)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e24.12 (29.35)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e35.23 (36.36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e52.70 (46.56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e66.59\u003c/p\u003e \u003cp\u003e(69.98)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSE \u0026plusmn;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e1.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCD 5%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e5.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e5.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCV (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e10.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e10.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e10.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e10.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"13\"\u003e\u003cem\u003eAll the treatments were significant at p-value\u003c/em\u003e\u0026thinsp;\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003e\u0026le;\u003c/span\u003e\u0026thinsp;\u003cem\u003e0.05 except before the spray\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eResidue Analysis\u003c/h2\u003e \u003cp\u003eResidue analysis of the Sarita Seedless grape variety under various treatments was conducted to determine the presence of multiple pesticides and their compliance with maximum residue limits (MRLs). Across all samples, residues of ethephon were observed in the range 0.010 to 0.363 mg/kg, remaining were below the MRL of 1.00 mg/kg. Residues of Di-thiocarbamates together assessed as CS\u003csub\u003e2,\u003c/sub\u003e varied from 0.191 to 0.639 mg/kg, in the allowed limit of 5.00 mg/kg. In buprofezin, residues were detected in most treatments, values ranged from 0.015 to 0.486 mg/kg, exceeding the allowable limit of 0.01 mg/kg in several cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Similarly, clothianidin showed 0.044 to 0.092 mg/kg, remaining below its limit of 0.70 mg/kg. The fungicide dimethomorph values showed 0.106 to 0.404 mg/kg, all within the allowable MRL of 3.00 mg/kg. The hexaconazole residual limit of 0.01 mg/kg was frequently detected, ranging between 0.010 and 0.022 mg/kg, often over and above the demarcated limit. For mandipropamid, residues limits were observed as 0.299 to 0.696 mg/kg, staying within the 2.00 mg/kg threshold. The fungicide metrafenone was present in concentrations between 0.392 and 0.654 mg/kg, below its MRL of 7.00 mg/kg in all samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Tebuconazole residues ranged from 0.015 to 0.352 mg/kg, within the MRL of 0.50 mg/kg, except in untreated controls. Additionally, 4-bromo-2-chlorophenol was detected in rootstock-treated samples at 0.183 mg/kg, above the standard limit of 0.01 mg/kg. Overall, most pesticide residues remained within their respective MRLs, indicating compliance with safety standards, except for buprofezin, hexaconazole, and 4-bromo-2-chlorophenol, which were found above permissible limits in specific treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe validated method demonstrated satisfactory linearity in the grape matrix, with correlation coefficients (r\u0026sup2;)\u0026thinsp;\u0026gt;\u0026thinsp;0.990. As per the result, the limit of quantification (LOQs) ranged from 0.004\u0026ndash;0.009 mg/kg, below the EU-established MRL for grapes. However, mean recoveries of selected pesticides were 78.40\u0026ndash;112.31%, with a percentage relative standard deviation (RSD) between 1.28\u0026ndash;6.85%, whereas, the matrix effects ranged from \u0026minus;\u0026thinsp;7.71\u0026ndash;23.2%, indicating minimal interference (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResidue analysis method validation recovery (%), and matrix effect (%) for selected pesticides in grape\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSr. No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eName of the compound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInstrument used for analysis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean % recovery at 0.010 mg/kg (n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e% RSD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMean % recovery at 0.050 mg/kg (n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e% RSD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMean % recovery at 0.100 mg/kg (n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e% RSD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eME (%) at 0.010 mg/kg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eLOQ at 0.010 mg/kg\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4-Bromo 2- chlorophenol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGC-MS/MS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e84.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e87.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e95.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e19.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.008\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBuprofezin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLC-MS/MS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e92.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e98.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e105.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e2.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-10.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eClothianidin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLC-MS/MS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e81.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e89.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e97.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e2.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-16.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDimethomorph\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLC-MS/MS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e88.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e85.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e95.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e2.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-5.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDithiocarbamates (Mancozeb, Maneb, Propineb, Metiram, Thiram, Zineb and Ziram collectively estimated as CS2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGC-MS/MS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e78.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e84.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e92.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e23.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.009\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEthephon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLC-MS/MS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e81.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e88.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e102.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e3.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-31.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.009\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEtrimfos\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLC-MS/MS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e79.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e83.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e89.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-28.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.008\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFluchloralin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGC-MS/MS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e83.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e80.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e91.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e3.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-21.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHexaconazole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLC-MS/MS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e95.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e105.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e110.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-9.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMandipropamid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLC-MS/MS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e91.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e96.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e103.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e2.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-13.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMetrafenone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLC-MS/MS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e94.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e99.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e106.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-11.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTebuconazole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLC-MS/MS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e98.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e107.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e112.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-7.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003eRSD: Relative standard deviation, ME: Matrix effect, LOQ: Limit of quantification\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe present study aimed to isolate the endophytic microbes, purify, characterize, develop their formulations, and evaluate their bio-efficacy against powdery mildew disease under field conditions in the grapevine cv. Sarita Seedless is a black-coloured table grape variety largely cultivated in the state of Maharashtra, India. The application of fungicides is limited for producing EU-MRL residue-compliant grapes. Hence, the aim was to manage disease by reducing the reliance on chemical fungicides and improving the quality and yield. The table and export grapes require minimal pesticide residues to meet consumer expectations, and the final harvest with the residual effect of chemicals is considered undesirable (Dumitriu et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Warneke et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). To align with the healthy produce, many vineyards adopt \"zero pesticides\" approach, thereby encouraging organic and natural practices (Roskaric et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This investigation assessed the efficient endophytic microbes to effectively manage powdery mildew disease. The impact of microbial inoculants on disease development was compared to sulphur-treated (positive control) and untreated plants. The chemical fungicides detected in the present study were applied before the flowering stage of the crop.\u003c/p\u003e \u003cp\u003eA comprehensive analysis was conducted to evaluate the treatment effects of PDI on foliage and berries, shelf-life, and loss in weight. Biochemical analyses of endophyte-treated grape samples included measurements of phenol, tannin, reducing sugars, proteins, proline, carbohydrates, and antioxidants. During both the crop seasons 2022-23 and 2023-24, all the isolates tested against grapevine showed excellent results on physical parameters of grapevine, including average berry weight, rachis length, total soluble solids (TSS), pH, berry length, and diameter, and other characteristics. In addition to this, the biochemical analysis of grapevine samples was estimated during both the years 2022-23 and 2023-24, which showed a positive impact on reducing sugar, tannin, protein, carbohydrates, and antioxidant properties of berries harvested from experimental plots. These findings were supported by the previous work evidenced that \u003cem\u003eTrichoderma\u003c/em\u003e application enhanced beneficial effects on TSS, pH, and acidity of grapevine crops (Csoto et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Carro-Huerga et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, \u003cem\u003eTrichoderma\u003c/em\u003e spp. not only increases the nutritional properties of the grapevine but it increases the accumulation of sugar in grapevine berries (Csoto et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, plants treated with endophytes consistently outperformed untreated controls across all experimental sites, and distinct reduction in the spread of disease in the treated vineyards. These findings were in accordance with the earlier reports on percent disease control with enhanced yield and quality (Bruisson et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Malviya et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Nasehi et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Efficacy of the fungal and bacterial endophytes could be achieved by various mechanisms, as evidenced from the recent research (Fadiji et al. 2020; Malviya et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), that led to inhibition of \u003cem\u003eE. necator\u003c/em\u003e in grapevine. Successful foliar sprays by these biocontrol agents indicate the strong rhizosphere and phyllosphere competence, a crucial factor for an effective biocontrol strategy (Santos et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Moreover, the endophytic \u003cem\u003eTrichoderma asperellum\u003c/em\u003e and \u003cem\u003eT. asprelloides\u003c/em\u003e could have supplemented the systemic resistance (ISR) in grapes. This mechanism protects against phytopathogens and environmental stressors (El-Sharkawy et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Pacifico et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Previous findings supported this investigation by Sawant et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), \u003cem\u003ein vivo\u003c/em\u003e applications of rhizospheric \u003cem\u003eTrichoderma\u003c/em\u003e developed ISR in grapes against the same pathogen. Previously, the efficiency of \u003cem\u003eBacillus\u003c/em\u003e spp. for powdery mildew has been extensively studied (Maachia et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Boiu-Sicuia et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Saha et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Holkar et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The EB strains \u003cem\u003eviz\u003c/em\u003e., SB5, RF1, SB2, and CS2 used in the present study were earlier assessed under laboratory conditions for direct and indirect antagonism for \u003cem\u003eC. gloeosporioides\u003c/em\u003e, the incitant of anthracnose in grapes in India (Holkar et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and found promising results. Moreover, field evaluations of these bioformulations for anthracnose disease are also in process.\u003c/p\u003e \u003cp\u003eIn the recent past, many research investigations have confirmed that the application of microbial formulations improved the nutritive quality parameters of grapevine and the health of plants simultaneously (Lombardi et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Verdenal et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Bettenfeld et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The investigation from this research supported quality parameters, but did not study the specific metabolites in fruits. The application of sulphur 80% WDG is an effective fungicidal strategy for powdery mildew disease management and to improve grapevine yield (Warneke et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Sulphur fungicide has protective properties rather than curative action. Therefore, during the present investigation, sulphur 80% WDG @ 2 g/L was used as a positive control. Sulphur strongly invades \u003cem\u003eErysiphe necator\u003c/em\u003e and prevents vineyards (Sellitto et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the vines treated with endophytic formulations, sulphur, and other fungicides were not used till the final harvest of the crop, except for the positive control. Therefore, the earlier bio-formulations of rhizospheric microbes were found efficient by integrating their application in combination with sulphur for enhanced disease management (Malviya et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This study significantly reduced disease on foliage and berries with improvement in post-harvest life when endophytic microbes\u0026rsquo; bio formulation was applied to the grapevine than the PC (S) and SDW treatments.\u003c/p\u003e \u003cp\u003eResidue analysis of cv. Sarita Seedless grapevine berries revealed that ethephon, di-thiocarbamates, clothianidin, dimethomorph, mandipropamid, metrafenone, and tebuconazole were all within the EU-MRL permissible limits. In the recent past, a strain of \u003cem\u003eEnterobacter\u003c/em\u003e spp. originated from the \u003cem\u003eMorus alba\u003c/em\u003e. Significantly reduced residues of Thiamethoxam in Chinese cabbage (Wang et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Five endophytic bacterial strains having plant growth-promoting activities degraded\u0026thinsp;\u0026gt;\u0026thinsp;90% of chlorpyrifos in rice plants and grains (Feng et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Moreover, in the recent past, Kumar et al. (2020) studied the biodiversity of endophytic microbial communities, including \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eTrichoderma\u003c/em\u003e species were found to degrade carbamate, organochlorine, organophosphate, and pyrethroids. In India, many endophytic microbes were isolated and found to have pesticide residue degradation ability in different crops and heavy metal detoxification (Mukherjee et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hazra et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shahid et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sharma et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Chemical residues were recently estimated from grape leaves infected by fungal diseases (Ailer et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In India, scant data are reported on pesticide residue management through endophytic microbes. Therefore, in the present study novel endophytic microorganisms were characterized for the management of powdery mildew disease and pesticide residues in the final harvest.\u003c/p\u003e \u003cp\u003eThe present formulations \u003cem\u003eviz\u003c/em\u003e., GB1, RF1, SB2, MCBY2, CSBY4, DRRS, and SB5 were found efficient in managing the disease and enhancing physiological and biochemical characteristics of the grape berries. Additionally, application of these bioformulations reduced the pesticide residues on the berry surface below the EU-MRL limits. This indicated that these grapevine endophytic microbes have multifaceted potential in grapes. Further, among these three bioformulations, \u003cem\u003eviz\u003c/em\u003e., MCBY2, DRRS, and SB5, were accepted for the multi-locational testing (MLTs). This information will be helpful for their registrations and legal usage in grapes. Moreover, combining all \u003cem\u003eTrichoderma\u003c/em\u003e bioformulations and \u003cem\u003eBacillus\u003c/em\u003e provided significant effects during 2023-24. This indicated that a consortium of \u003cem\u003eTrichoderma\u003c/em\u003e isolates is needed for devising an efficient disease management strategy in grapevine.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe present study demonstrated the impact of endophytic microbial formulations of \u003cem\u003eTrichoderma\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e species in managing powdery mildew disease in grapevines while improving the physical, biochemical, and shelf-life parameters of grape berries. Residue analysis indicated compliance with EU-MRLs for most of the pesticides, ensuring safety and suitability for consumption and export. The study underscores the potential of integrating endophytic microbes into sustainable farming practices, offering an eco-friendly, safe and optional strategy for chemicals in disease management, especially when fungicide application at berry development stage is not possible.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare that they have no conflict of interest in the publication.\u003c/p\u003e\n\u003cp\u003eSKH: conceived ideas and formulated the experiments, drafted and reviewed the manuscript, SCN: isolated, characterized, and evaluated the bioformulations of endophytes under field conditions and prepared tables, SBB: analysed sequence information and constructed phylogenetic trees, SJP: prepared the figures and reviewed the manuscript, HNM: reviewed and drafted the manuscript, BST: residue analysis of the grape samples, SDG: Statistical analysis, NAD: reviewed and drafted the manuscript, KB: critically reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccession Numbers\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOQ407830, OQ402731, OQ781200, OQ503170, OQ784170, OM280451, OL778931, OM280455, OM341599, OL797982, ON063017, OQ473591\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors highly acknowledge the support provided by the Director, ICAR-National\u003c/p\u003e\n\u003cp\u003eResearch Centre, Pune to carry out the proposed research work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAiler S, Benesova L, Baron M, Galovicova L (2024) Monitoring of pesticide residues in grapevine leaves under variant fungal disease management. 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Nanosci Nanotech Lett 8:532\u0026ndash;538. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1166/nnl.2016.2164\u003c/span\u003e\u003cspan address=\"10.1166/nnl.2016.2164\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biocontrol, grapes, powdery mildew, endophytes, Trichoderma asperellum, Bacillus subtilis","lastPublishedDoi":"10.21203/rs.3.rs-6602877/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6602877/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGrapevine (\u003cem\u003eVitis vinifera\u003c/em\u003e L.) plays a major role in producing table grapes, raisins, juice, and wine in the global market. In the present study, 13 endophytes were isolated, purified, and identified by sequencing the ITS and 16S rRNA regions. Based on ITS sequence information, the fungal endophytes \u003cem\u003eviz.\u003c/em\u003e, CSBY-2, CSBY-8, and MCBY-2, were identified as \u003cem\u003eTrichoderma asperellum\u003c/em\u003e, whereas CSBY-4, MCBY-1, and DRRS-1 were identified as \u003cem\u003eT. asperelloides\u003c/em\u003e, \u003cem\u003eCytospora\u003c/em\u003e sp., and \u003cem\u003eT. viride\u003c/em\u003e, respectively. Similarly, the bacterial endophytes \u003cem\u003eviz\u003c/em\u003e., Sauvignon Blanc-2, -5, CS2, and RF1, were characterized as \u003cem\u003eBacillus subtilis\u003c/em\u003e. The RT7 and RB-1 were recognized as \u003cem\u003eB. mojavensis\u003c/em\u003e and \u003cem\u003eB. licheniformis\u003c/em\u003e, respectively, by sequencing 16S rRNA. The fungal endophytes were formulated into powder using flattened rice as a carrier, and bacterial isolates were used as liquid formulations. Bioformulations of these isolates were evaluated against grapevine powdery mildew disease during the 2022-23 and 2023-24 crop seasons at the experimental fields at ICAR-NRCG, Pune. In field evaluation of endophytic \u003cem\u003eTrichoderma\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eCytospora\u003c/em\u003e species formulations during the 2022-23 and 2023-24 crop seasons, the highest percent disease control (PDC) on leaves was recorded in treatment, \u003cem\u003eBacillus subtilis\u003c/em\u003e (RF-1: 63.41%) followed by \u003cem\u003eB. subtilis\u003c/em\u003e (SB2: 61.57% and 58.11%), \u003cem\u003eBacillus\u003c/em\u003e sp. (GB2: 61.04%), \u003cem\u003eB. subtilis\u003c/em\u003e (SB5: 59.66% and 56.65%), and \u003cem\u003eTrichoderma asperellum\u003c/em\u003e (MCBY2: 51.07% and 60.93%). The highest PDC on bunches was observed in SB2 (64.56% and 44.97%), SB5: (56.18% and 46.07%), and MCBY-2: 55.88% and 67.7%) isolates. Moreover, all the fungal and bacterial formulations reduced the fungicide residues in the grape berries. The study provides important insights into managing grapevine diseases sustainably and suggests integrating endophytic microbes into traditional farming practices to enhance grape quality and productivity.\u003c/p\u003e","manuscriptTitle":"Characterization of grapevine endophytic microbes and field evaluation of bioformulations against powdery mildew disease with residue-compliant grape production ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-16 16:40:06","doi":"10.21203/rs.3.rs-6602877/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4cee0c5e-deaa-4d8c-bca6-9b2eb59bde8d","owner":[],"postedDate":"May 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-16T09:09:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-16 16:40:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6602877","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6602877","identity":"rs-6602877","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}