Cross-kingdom microbial Consortiums efficiently Degradate Tomato Root litter

Cross-kingdom microbial Consortiums efficiently Degradate Tomato Root litter | 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 Cross-kingdom microbial Consortiums efficiently Degradate Tomato Root litter Yanhui Wang, Rui Wang, Yanan Xue, Dandan Pan, Jingyu Zhang, Fengzhi Wu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9101939/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract The accumulation of root litter in soil can cause soil sickness, which negatively affect crop production. Here, we isolated bacterial and fungal strains capable of degrading tomato root litter and construct efficient cross-kingdom microbial consortium. Through in vitro screening, we obtained 16 strains, including five bacterial strains and eleven fungal strains that can degrade tomato root litter. The degradation abilities of the fungal strains were generally higher than that of the bacterial strains. Then, we successfully contrasted two cross-kingdom microbial consortiums Z2 (containing Aspergillus sp., Sarocladium strictum , Cellulosimicrobium funkei , and Penicillium sp.) and consortium Z3 (containing Aspergillus sp., Sarocladium strictum , Cellulosimicrobium funkei , and Microbacterium sp.) that could efficiently degrade tomato root litter. The microbial strains in each consortium were compatible to each other. These two microbial consortia showed higher degrading efficiency than any single strain in each consortium. The results indicate that fungi play a dominant role in the degradation of tomato root litters, and certain compatible strain consortia can enhance the degradation capacity. Tomato root litter Strain screening Microbial degradation Strain compatibility Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Tomato is an important cultivated vegetable in China, with both planting area and yield ranking among the forefront globally. Highly intensive production has become a characteristic of modern agriculture. However, the gradual simplification of crop species in protected cultivation, along with continuous monocropping of the same vegetable on the same land year after year, has led to a decline in production quality and an increase in pests and diseases. This cultivation practice negatively affects the sustainable production of crops (Wu and Zhou 2009 ). Research indicates that the causes of continuous cropping obstacles can be divided into three main categories: deterioration of soil physicochemical properties, and root exudates coupled with the decomposition of root litters (Uehling, Entler et al. 2019). Among these, the accumulation of toxic substances and the imbalance of soil microbial communities are significant contributing factors.Following continuous cropping, root litters persist in the soil. While their decomposition can release nutrients, enhance levels of soil organic carbon, phosphorus, and potassium, and thereby improve soil physicochemical properties,the degradation process is in fact slow (Fu, Chen et al. 2021). In the short term, microbial activity often triggers a "nitrogen competition" effect, leading to nitrogen immobilization (Chen, Liu et al. 2014). Furthermore, the accumulated litters attract pathogen colonization and reduce populations of beneficial microorganisms, disrupting the balance of the soil microbial community and contributing to disease outbreaks (Wu, Jiao et al. 2017). For instance, common soil-borne diseases in continuous tomato cropping, such as Fusarium wilt and bacterial wilt, are primarily caused by the buildup of Fusarium oxysporum and Ralstonia solanacearum in the soil (Basco, Bisen et al. 2017, Yin, Zhang et al. 2022 ). Additionally, During decomposition, root litters also release phenolic allelochemicals such as coumaric acid and ferulic acid, which reduce soil pH and organic matter content, thus altering the soil's physicochemical properties (Wang, Yang et al. 2024). Although returning crop litters to the field offers certain agronomic benefits, these are frequently outweighed by the severe drawbacks induced by continuous cropping obstacles, which are detrimental to long-term sustainability. Therefore, accelerating the degradation of root litters represents a crucial measure for mitigating these obstacles. Microorganisms serve as decomposers in ecosystems and play a vital role in maintaining ecological balance. Due to their environmentally friendly and non-toxic degradation processes, microbial inoculants have been widely adopted (Sruthy, Shukla et al. 2023). Numerous studies have demonstrated the ability of microorganisms to degrade root litters and secondary metabolites. Many saprophytic fungi and Streptomyces species can secrete various extracellular enzymes to break down soluble substances and lignin (Zhou, Fransen et al. 2025), Certain Penicillium species utilize phenolic acids as a carbon source, directly decomposing toxic substances. Various microorganisms not only exhibit litter-degrading capabilities but also demonstrate additional beneficial traits. For instance, the combined application of Bacillus subtilis with humic acid and chitosan can suppress the abundance of pathogens in continuous tomato cropping soil, reduce phenolic acid content, and mitigate disease incidence (Qiu, Bao et al. 2025).Additionally, certain microorganisms can enhance plant disease resistance and exhibit growth-promoting properties. For instance, Streptomyces sp. SK68 not only demonstrates multiple degrading enzyme activities but also increases tomato biomass and alleviates salt stress after inoculation (Damodharan, Palaniyandi et al. 2018).While many microorganisms show some effect in degrading root litters, a large proportion demonstrate low efficiency. Therefore, it is necessary to screen for highly efficient strains capable of degrading tomato root litters. In this study, we aimed to isolate and purify microbial strains from tomato root litters degraded for 15 and 60 days. Microorganisms capable of degrading various components of the litters were screened, and strains with high degradation activity were selected based on enzyme assays in liquid enzyme‑production media.The degradation capabilities of the screened strains and their combinations were further evaluated through in vitro root litter degradation assays. This work aims to provide effective strain resources for the subsequent development of bioagents and to offer a theoretical basis for alleviating tomato continuous cropping obstacles. Materials and methods Root litter Degradation Experiment We collected tomato root litters from continuous cropping soil. These litters were rinsed clean, dried until constant weight was achieved, and then crushed into small segments of 1–2 cm. Continuous cropping tomato soil was collected in May 2018 from the experimental station of Northeast Agricultural University in Harbin, China (45°41′N, 126°37′E).1 g of tomato root litters was mixed with 100 g of continuous cropping soil in 250 ml culture flasks. The soil was used to cover and homogenize the litters, and soil moisture was maintained at 65% of the field water-holding capacity. Each sampling time point included three replicates, with four flasks per replicate, totaling 24 flasks. All flasks were incubated in the dark at 25 ± 1°C. Root litters were sampled at 15 days and 60 days for subsequent screening of tomato litter-degrading microorganisms. Strain Isolation After rinsing off soil particles attached to the tomato root litters with sterile water and surface‑sterilizing, the root litters were fully ground using PBS standard buffer under aseptic conditions. The ground mixture was transferred into a sterilized conical flask to obtain a suspension, which was then serially diluted to concentration gradients of 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, and 10⁻⁷. A 200 µL aliquot of each dilution was spread onto Beef Extract Peptone Agar and Rose Bengal Agar for the isolation of bacteria and fungi, respectively. The plates were inverted and incubated in a constant-temperature incubator (Bacteria: 37°C for 24 hours; Fungi: 30°C). After colony formation, individual colonies were picked and streaked onto LB agar (for bacteria) or PDA agar (for fungi) to obtain pure isolates. All isolated bacteria and fungi were further purified on LB and PDA media, respectively. After culturing for 3–5 days, the strains were stored at 4°C and subsequently inoculated onto various screening media for functional screening in the short term. Screening for Root litter-Degrading Microorganisms The isolated strains were inoculated onto a soluble starch medium (composition: ammonium sulfate 0.5%, K₂HPO₄ 0.5%, soluble starch 0.5%, peptone 1.5%, beef extract 0.3%, agar 0.8%, distilled water, pH 7.0). After inoculation, the plates were inverted and incubated in a constant-temperature incubator (bacteria: 37°C for 24 hours; fungi: 30°C for 2 days). Iodine solution was then dropped onto the developed colonies, and single colonies surrounded by starch hydrolysis halos were selected. Strains forming larger clear halos were chosen for secondary screening (Ogbonnaya and Odiase 2012 ). Milk medium plates (composition: beef extract 0.3%, peptone 1%, NaCl 0.5%, agar 2%, pH 7.4–7.6,supplemented with 1.5% autoclaved skim milk) were used for screening protein‑degrading strains. Inoculation was performed as described above (Ma, Li et al. 2024)(bacteria: 37°C for 24 h; fungi: 30°C for 3 days). Strains capable of forming clear zones were selected, and those producing larger halos were subjected to secondary screening. Pectin primary screening medium (composition: K₂HPO₄ 0.1%, MgSO₄ 0.05%, NaNO₃ 0.3%, FeSO₄ 0.001%, pectin 0.2%, agar 1.5%, pH 7.0) was used to screen for pectin‑degrading strains. After incubation, Congo Red solution (0.2%, w/v) was added to the plates and allowed to stain for 2 h. The dye solution was then discarded, and the plates were washed with NaCl (1 M) for destaining (KC, Upadhyaya et al. 2020). Strains forming larger clear zones were similarly selected for secondary screening. Agar CMC‑Na medium (composition: CMC‑Na 1.5%, NH₄NO₃ 0.1%, yeast extract 0.1%, MgSO₄·7H₂O 0.5%, K₂HPO₄ 0.1%, agar 1.5%) was used for screening cellulose‑degrading strains (Wei, Pan et al. 2004). After incubation, Congo Red solution (0.1%, w/v) was applied for 30 min. The dye was discarded, and the plates were destained with NaCl (1 M). Strains producing larger clear zones were chosen for secondary screening. GU-PDA medium (PDA supplemented with 0.1% guaiacol) and Azure B medium (composition: yeast extract 1%, glucose 1%, Azure B 0.01%, agar 2%) were used for screening lignin-degrading strains. Incubation was carried out under the following conditions: bacteria at 37°C for 2 days; fungi at 28°C for 7 days. Changes in colony color and morphology were observed. Strains producing brown colored halos or clear degradation zones were selected for secondary screening, respectively (Bibi and Bhatti). Rescreening of Strains The selected bacterial and fungal strains were inoculated into LB liquid medium and PDB medium, respectively, and cultured with shaking at 30°C and 170 rpm. When the bacterial OD600 or fungal spore concentration reached approximately 10⁷ CFU/mL, the cultures were inoculated at 5% (v/v) inoculum size into liquid enzyme‑production medium.Each treatment was performed in triplicate and incubated with shaking at a constant 30°C. Samples were taken on days 2, 4, 6, and 8. After collection, the samples were transferred to 50 mL centrifuge tubes and centrifuged at 10,000 rpm for 10 min.The supernatant (the crude enzyme extract) was collected for subsequent enzyme activity assays. Crude enzyme extracts for starch, protein, pectin, and cellulose degradation were prepared using the method described above. For lignin-degrading enzyme production, 0.5 g of root litters was placed into a 50 mL conical flask, followed by the addition of 10 mL of HNHC liquid medium (composition: glucose 2%, ammonium tartrate 0.2%, K₂HPO₄ 0.1%, MgSO₄·7H₂O 0.05%, CuSO₄·5H₂O 0.001%, ZnSO₄·7H₂O 0.0005%, MnSO₄ 0.0005%, pH 6.0). The flasks were autoclaved(Baldrian and Snajdr 2006 ). A 5 mm mycelial plug taken from the edge of a seven‑day‑old fungal colony grown on a plate was inoculated into each flask. For bacterial strains, 1 mL of a bacterial suspension at 10⁷ CFU/mL was added to each flask. Sampling was performed on days 7, 9, 11, 13, and 15. At each sampling point, 10 mL of sterile water was added to each flask, followed by homogenization at 4°C and 150 rpm for 2 h. The mixture was then transferred to 50 mL centrifuge tubes and centrifuged to obtain the crude lignin-degrading enzyme solution. Amylase activity was determined using the DNS method. The reaction mixture was heated in a boiling water bath for 10 min and then cooled (Mishra and Behera 2008 ). The optical density of each sample and the reaction mixture was measured at 575 nm using a microplate reader. One unit of enzyme activity was defined as the amount of enzyme that released 1 µmol of glucose per minute under the assay conditions. Protease activity was measured by the Folin-phenol method (Zhao, Wang et al. 2016). The optical density of the reaction mixture was measured at 680 nm using a microplate reader. One unit of enzyme activity was defined as the amount of enzyme required to produce 1 µmol of tyrosine per minute. Pectinase activity was also assayed using the DNS method. A 1% pectin solution was prepared in 0.2 M sodium acetate buffer (pH 5.5). Then, 0.2 mL of crude enzyme extract was mixed with 0.8 mL of the substrate and incubated at 50°C for 1 h. The reaction was terminated by adding 1.5 mL of DNS reagent, followed by heating in a boiling water bath for 10 min. The volume was adjusted to 9 mL, and absorbance was measured at 540 nm using a microplate reader. One unit of enzyme activity was defined as the amount of enzyme that released 1 µmol of reducing sugar (galacturonic acid equivalent) per minute. Cellulase activity was determined by the DNS method. A 1% sodium carboxymethyl cellulose solution was prepared in 0.2 M sodium acetate buffer (pH 5.0). Then, 0.1 mL of crude enzyme extract was mixed with 1.9 mL of the substrate and incubated at 50°C for 20 min. The reaction was terminated by adding 1.5 mL of DNS solution, followed by heating in a boiling water bath for 10 min. After cooling, the absorbance was measured at 520 nm using a microplate reader. One unit of enzyme activity was defined as the amount of enzyme required to produce 1 µg of glucose per minute. Lignin-degrading enzyme activities were determined separately for two enzymes: laccase was assayed using the ABTS method, Peroxidase activity was determined by the veratryl alcohol method. (Li, Zhu et al. 2014). Strain Identification Genomic DNA of bacterial and fungal strains was extracted using bacterial and fungal genomic DNA extraction kits, respectively. For fungi, the ITS region of the rRNA gene was amplified using the primers ITS1/ITS4 (Henry, Iwen et al. 2000). For bacteria, the 16S rRNA gene sequence was amplified using the primers 27F/1492R. All amplified products were sequenced. The obtained ITS and 16S rRNA gene sequences were compared with those of other strains in the GenBank database using the NCBI BLASTn tool. Phylogenetic trees were constructed using the neighbor-joining method in MEGA 11.0 software. The stability of the tree clades was assessed with bootstrap values based on 1000 replicates. Determination of Strain Compatibility and Degradation Efficiency Compatibility tests were conducted between highly efficient strains capable of degrading different components of root litters. The compatibility between fungal strains, as well as between fungal and bacterial strains, was assessed using the dual‑culture plate confrontation method (Santiago, Yagi et al. 2017). The compatibility between bacterial strains was determined using the cross-streak method. Based on these results, compatible strains were selected to establish microbial consortia designated as Z1, Z2, Z3. To determine the degradation efficiency of individual strains and consortia, 2 g of root litters were weighed and placed into 100 mL conical flasks. Each flask was supplemented with 20 mL of inorganic salt medium to submerge the litters. After sealing with sealing film and autoclaving, 2 mL of a highly efficient tomato root litter‑degrading bacterial suspension (concentration: 10⁷ CFU/mL) was inoculated into each flask. Each treatment was performed in triplicate. Individual strains and the constructed consortia served as experimental groups. A control group was set up by adding the same volume of magnesium sulfate solution without bacterial inoculation. All flasks were incubated in the dark at 30°C. Samples were taken on day 15(Li, Wang et al. 2011, Yang, Wang et al. 2017). The root litters were rinsed with sterile water, oven‑dried at 80°C, and weighed. The degradation rate of tomato root litters was calculated using the weight‑loss method: Degradation rate (%) = [(m₀ – m) / m₀] × 100%, where m₀ is the initial weight of the root litters and m is the weight of the root litters after rinsing and drying on day 15 following inoculation (Zhang, Wang et al. 2022). Statistical analysis All experimental data were analyzed using one-way analysis of variance (one‑way ANOVA), followed by multiple comparisons with the Tukey HSD test. The significance level was set at P < 0.05. All bar charts and line graphs were generated using Origin Pro 8.5. Results Screening of Root litter-Degrading Microorganisms Primary screening results showed that 477 bacterial and 417 fungal with degradation ability were obtained from the 15‑day degraded root litters, while 713 bacterial and 408 fungal were isolated from the 60‑day degraded litters. Larger degradation halos indicate stronger degradation ability. Strains exhibiting larger halos were therefore selected for rescreening. Rescreening of Strains Based on the primary screening results, 16 strains exhibiting degrading enzyme activities were selected. Among them, the fungal strain Simplicillium sp. bb60‑73 showed the highest amylase activity, reaching 6.8 U/mL on day 2 (Figure.1a). The highest protease activity was observed in the bacterial strain Paenibacillus sp. ab60‑72, which remained stable between 4.0 and 4.5 U/mL (Figure.1b). For pectinase, the fungal strain Albifimbria verrucaria bc15-20 exhibited the highest activity, peaking at 4.4 U/mL on day 2 (Figure.1c). The bacterial strain Paenibacillus sp. bc60‑90 displayed the highest cellulase activity, reaching 1.55 U/mL on day 8 (Figure.1d). Regarding lignin‑degrading enzymes, the highest laccase activity was recorded for the fungal strain Trametes sp. ab60‑59 (2.2 U/mL) (Figure.1e), and the highest peroxidase activity for the fungal strain Penicillium sp. ca60‑13 (4.56 U/mL) (Figure.1f), both peaking on day 15. Identification of Degrading Strains The 16S rRNA and ITS1 gene sequences of the 16 selected degrading strains were obtained for homology analysis. Sequences with similarity above 99% were selected to construct a phylogenetic tree using MEGA 11.0. Strains ca60‑52(PX970998A) and ba60‑13(PX970997A) were preliminarily identified as bacteria belonging to the genus Microbacterium ; strain bb60‑111(PX964323A) was identified as Cellulosimicrobium funkei (Fig. 2 ). strains bb60‑72(PX964323A) and bc60‑90(PX964330A) were assigned to the genus Paenibacillus . Among the fungal strains, bc60‑61(PX963975A), aa60‑12(PX963889A), and ac60‑80(PX963974A) were identified as members of the genus Penicillium ; ca60‑51(PX963842A) and bc60‑63(PX964219A) were identified as Aspergillus sp; ab60‑73(PX963963A) was identified as Simplicillium sp.; bb60‑2(PX963973A) was identified as Sarocladium strictum ; cb60‑55(PX964257A) and bc15-20(PX975913) was identified as Paramyrothecium roridum ; ab60‑59(PX963890A) was preliminarily identified as a fungus of the genus Trametes ; and ca15‑65(PX964223A) was identified as a Fusarium sp (Fig. 3 ). The 16S rDNA and ITS1 gene sequences of the strains were submitted to GenBank to obtain the sequence accession numbers. Determination of Strain Compatibility and Degradation Rate Pairwise compatibility tests among the 16 degrading strains revealed that fungal strain aa60‑12 was incompatible with fungal strains bc15-20 and ab60‑73, while all other strain combinations were compatible, Based on the compatibility results, strains were selected from the compatible isolates and used to construct microbial consortia. (Fig. 4 ). The 16 screened degrading strains were subjected to an in vitro root litter degradation test. On day 15, fungal strain bc60‑61 showed the highest degradation rate (39.56%), while bacterial strain bb60‑72 exhibited the lowest (11.33%). With the exception of fungal strain aa60‑12, all other fungal strains demonstrated greater degradation efficiency than the bacterial strains (Fig. 5 ). In contrast, compatible consortia achieved the following degradation rates: Z1 (bc15-20 and ab60‑73) 26.56%, Z2 (bc60-63, bb60‑2, bb60-111, and aa60-12) 33.49%, and Z3 (bc60‑63, bb60‑2, bb60-111, and ba60-13) 34.18%.Among these, the degradation rate of consortium Z1 was comparable to that of its constituent single strains. Consortia Z2 and Z3 exhibited significantly higher degradation rates than any individual strain within the respective groups, demonstrating a pronounced synergistic effect on tomato root litter degradation. Discussion In this study, microorganisms were isolated and purified from tomato root litters degraded for 15 and 60 days using the dilution plating method. It was observed that as the degradation period extended, the number of bacterial colonies decreased, while the number of fungal colonies increased. This phenomenon may be attributed to the faster growth rate of bacteria during the early stages of decomposition, allowing them to preferentially utilize available nutrients (Rousk, Brookes et al. 2010).In contrast, fungi, despite their slower initial growth, are capable of secreting a wider array of enzymes that degrade recalcitrant substances in the later phases of decomposition (Lorenz and Lal 2005 , Poll, Marhan et al. 2008). These findings are consistent with the results obtained in our experiments. The in vitro root litter degradation assay revealed that fungi overall exhibited stronger degradation capabilities compared to bacteria. Among them, the Penicillium strain bc60‑61 showed the highest degradation ability. Fungi from the genera Simplicillium and Aspergillus were not only the most abundant but also demonstrated strong degradation performance (Hahn Schneider, Goncalves et al. 2016). Basidiomycete strains such as bc15-20 and cb60‑55 also possessed relatively high degradation abilities for cellulose and lignin. This may be attributed to the more comprehensive repertoire of enzymes secreted by fungi, enabling them to effectively degrade lignin—the primary component of root litters.The fungi isolated in this study predominantly belonged to Ascomycota, which is consistent with reports indicating that Ascomycota fungi play a dominant role during the early to middle stages of litter degradation (Deacon, Pryce-Miller et al. 2006). The Penicillium and Aspergillus strains screened here exhibited strong root litter degradation ability, with bc60‑61 performing the best. This high efficiency may be attributed to the secretion of various hydrolytic enzymes and antibiotics by these fungal genera, which can suppress competing microorganisms, including pathogens, thereby facilitating their dominance in the decomposition process (Hiscox and Boddy 2017 ). Some of the screened strains belonged to the genus Fusarium , which is frequently associated with tomato wilt diseases. However, whether strain ca15‑65 is pathogenic remains unclear and requires further experimental verification.Among the bacterial isolates, strains from the phylum Actinobacteria demonstrated relatively strong degradation capabilities. Strains bb60‑111 and ca60‑52, all belonging to Actinobacteria, exhibited higher degradation activity than those from the phylum Firmicutes.Notably, strain ba60‑13 also showed the ability to degrade cellulose and was the most efficient bacterial strain in degrading root litters. This can be attributed to the capacity of Actinobacteria to secrete a variety of hydrolytic enzymes, endowing them with strong in vitro degradation potential for plant litters (Bao, Dolfing et al. 2021). Studies have shown that microorganisms can interact and communicate with each other, and the construction of microbial communities can enhance the degradation ability of root litters (Sruthy, Shukla et al. 2023, Singh, Abiraami et al. 2025). In this study, compatibility tests were conducted among the 16 screened strains. The compatible consortium Z1, consisting of bc15-20 and ab60‑73, did not exhibit a significant improvement in degradation rate, suggesting that these two strains neither strongly synergize nor inhibit each other under the tested conditions. Consortia Z2 and Z3 demonstrated significantly enhanced degradation capabilities compared to their individual constituent strains, indicating that compatible multi‑species combinations improve degradation efficiency. Fungi can efficiently decompose root litters, and the intermediate products are fully utilized by bacteria (Zhang, Wen et al. 2025), creating a synergistic relationship. Moreover, fungi can secrete compounds that promote bacterial growth (Uehling, Entler et al. 2019). Increased microbial diversity also strengthens the stability of the community, and the various metabolites produced can directly or indirectly suppress pathogen spread and proliferation (Singh, Jiang et al. 2025). Similarly, combinations of four Firmicutes bacteria enhanced plant immune responses and improved tomato resistance compared to single or paired bacterial treatments (Lee, Kong et al. 2021). Conclusion In this study, using tomato root litters degraded for 15 and 60 days as materials, 11 fungal strains and 5 bacterial strains capable of degrading root litter components were isolated. The degradation ability of fungi was generally higher than that of bacteria, with fungi from genera such as Penicillium , Aspergillus , Paecilomyces , and Fusarium showing a more pronounced advantage. Strain compatibility assays revealed that certain compatible consortia enhanced the degradation efficiency of root litters. This work lays a foundation for subsequently constructing microbial consortia for tomato root litter degradation. Declarations Acknowledgements This research was supported by the National Natural Science Foundation of China [32573013, 32402556]. Disclosure statement No potential conflict of interest was reported by the author(s). References Baldrian P, Snajdr J (2006) Production of ligninolytic enzymes by litter-decomposing fungi and their ability to decolorize synthetic dyes. Enzym Microb Technol 39(5):1023–1029 Bao Y et al (2021) Important ecophysiological roles of non-dominant Actinobacteria in plant residue decomposition, especially in less fertile soils. Microbiome 9(1) Basco MJ et al (2017) Biological management of Fusarium wilt of tomato using biofortified vermicompost. Mycosphere 8(3):467–483 Bibi I and H. N. 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World J Microbiol Biotechnol 33(7) Yang CX et al (2017) Isolation, identification and characterization of lignin-degrading bacteria from Qinling, China. J Appl Microbiol 123(6):1447–1460 Yin J et al (2022) Heritability of tomato rhizobacteria resistant to Ralstonia solanacearum. Microbiome 10(1) Zhang W et al (2025) The Fungi-Bacteria Interaction Mechanism of Microbial Consortium During Efficient Lignin Degradation Based on Metabolomics Analysis. Molecules 30(3) Zhang X et al (2022) The Influence of Residue Mixing on the Decomposition of Pepper Root Residues. 12(1):84 Zhao R et al (2016) Bioremediation of Hexavalent Chromium Pollution by Sporosarcina saromensis M52 Isolated from Offshore Sediments in Xiamen, China. Biomed Environ Sci 29(2):127–136 Zhou Q et al (2025) Lignin-Degrading Enzymes and the Potential of Pseudomonas putida as a Cell Factory for Lignin Degradation and Valorization. 13(4): 935 Additional Declarations No competing interests reported. Supplementary Files strainsequence.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 16 Apr, 2026 Editor assigned by journal 14 Mar, 2026 Submission checks completed at journal 14 Mar, 2026 First submitted to journal 12 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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-9101939","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":626947374,"identity":"5bc748a4-6bdd-4762-93cf-6c371b7ea553","order_by":0,"name":"Yanhui Wang","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yanhui","middleName":"","lastName":"Wang","suffix":""},{"id":626947375,"identity":"0b7e8193-565b-4e75-8729-75744ef40d9b","order_by":1,"name":"Rui Wang","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Wang","suffix":""},{"id":626947377,"identity":"e52ace29-b8b5-432b-b528-a61324eceb4c","order_by":2,"name":"Yanan Xue","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yanan","middleName":"","lastName":"Xue","suffix":""},{"id":626947380,"identity":"bc811e26-f47a-4255-a783-2e33cb1a8cf5","order_by":3,"name":"Dandan Pan","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Dandan","middleName":"","lastName":"Pan","suffix":""},{"id":626947382,"identity":"e3ad27fc-860a-4595-aa09-e6d45773076e","order_by":4,"name":"Jingyu Zhang","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jingyu","middleName":"","lastName":"Zhang","suffix":""},{"id":626947388,"identity":"d90c49b6-118b-4379-863a-67ebec0c52d3","order_by":5,"name":"Fengzhi Wu","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Fengzhi","middleName":"","lastName":"Wu","suffix":""},{"id":626947389,"identity":"b360f5d4-ee6f-486a-888d-eba7491aeb48","order_by":6,"name":"Xingang Zhou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYHACNiC2QWITqSWNdC2HSdBicH75swcf287n8bOfMWD4UHaYgX92A34tkjMepBvObLtdLNmTY8A449xhBok7B/Br4Zc4cEyat+124oYDOQbMvG2HGQwkEgh4ROJgG1DLucT9598YMP8lRgs/fzMbUMuBxA0SQFsYidEiOYONTXLGueTEGTeeFRzsOZfOI3GDgBaD88efSXwos0vs70/e+OBHmbUc/wwCWhiQnXEAiHkIqAcC/gOE1YyCUTAKRsEIBwBpBEJ0o8lZgQAAAABJRU5ErkJggg==","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Xingang","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2026-03-12 08:09:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9101939/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9101939/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107706729,"identity":"91b89dd0-db3c-4a5b-aee4-3cd8bb90af6b","added_by":"auto","created_at":"2026-04-24 09:18:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":258751,"visible":true,"origin":"","legend":"\u003cp\u003e(a)Enzyme activity assay of starch-degrading strains. (b)Enzyme activity assay of protein-degrading strains. (c)Enzyme activity assay of pectin-degrading strains. (d) Enzyme activity assay of lignin-degrading strains. (e-f)Enzyme activity assay of cellulose-degrading strains.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9101939/v1/127071ca179a3ae673450173.png"},{"id":107707612,"identity":"52d271e3-9052-4872-90a5-59f6d29bb30a","added_by":"auto","created_at":"2026-04-24 09:20:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":199221,"visible":true,"origin":"","legend":"\u003cp\u003eNeighbor-joining tree showing the the 16S RNA region of bacterial rRNA genes sequence of 5 fungal strains. Colony morphology of 5 root litter-degrading bacterial strains on LB media. Only bootstrap values above 70% (percentages of 1000 replications) are indicated. Bar, 10 nucleotide substitutions per site\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9101939/v1/c9edbb2397006dbab9b2b101.png"},{"id":107674726,"identity":"d9987bea-c648-4ae0-96a4-ad93f89339db","added_by":"auto","created_at":"2026-04-24 00:24:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":364735,"visible":true,"origin":"","legend":"\u003cp\u003eNeighbor-joining tree showing the the ITS1 region of fungal rRNA genes sequence of 11 fungal strains. Colony morphology of 11 root litter-degrading fungal strains on PDA media. Only bootstrap values above 70% (percentages of 1000 replications) are indicated. Bar, 20 nucleotide substitutions per site.\u003cimg width=\"69\" height=\"35\" src=\"file:///C:/Users/kamb13/AppData/Local/Temp/msohtmlclip1/01/clip_image001.gif\" alt=\"Text Box: ac60-80 \"/\u003e\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9101939/v1/ee0763fb32eea56f38d5fedb.png"},{"id":107674728,"identity":"62e10bc4-a0d4-4ead-8edd-f6447b190c88","added_by":"auto","created_at":"2026-04-24 00:25:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":398952,"visible":true,"origin":"","legend":"\u003cp\u003eFungal Compatibility with Bacteria and Other Fungi. Z1:compatible strains bc15-20 and ab60-73, Z2:aa60-12, bb60-111, bc60-63, bb60-2, Z3:ba60-13, bb60-111, bc60-63, bb60-2\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9101939/v1/c1f1d2467b0c234bc0f59e51.png"},{"id":107674725,"identity":"832863bc-c872-4f2e-803e-f46cf4d2f6d7","added_by":"auto","created_at":"2026-04-24 00:24:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":597128,"visible":true,"origin":"","legend":"\u003cp\u003eDegradation rate of root litters by individual strains and Strain Combinations. Control:. Z1: compatible strains bc15-20 and ab60-73. Z2:ompatible strains (aa60-12, bb60-111, bc60-63, bb60-2). Z3:compatible strains (ba60-13, bb60-111, bc60-63, bb60-2).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9101939/v1/061fd1ed9c9bf69804d33ffb.png"},{"id":107709215,"identity":"a24ad7de-49a2-4e88-9d28-6cece473c1fd","added_by":"auto","created_at":"2026-04-24 09:35:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2031393,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9101939/v1/7dd5e167-ec3f-4dac-8362-14722af47af5.pdf"},{"id":107674723,"identity":"249ebf05-5191-4d21-9800-43d70a90aa7c","added_by":"auto","created_at":"2026-04-24 00:24:59","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":15940,"visible":true,"origin":"","legend":"","description":"","filename":"strainsequence.docx","url":"https://assets-eu.researchsquare.com/files/rs-9101939/v1/611265f721d15847b24be858.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cross-kingdom microbial Consortiums efficiently Degradate Tomato Root litter","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTomato is an important cultivated vegetable in China, with both planting area and yield ranking among the forefront globally. Highly intensive production has become a characteristic of modern agriculture. However, the gradual simplification of crop species in protected cultivation, along with continuous monocropping of the same vegetable on the same land year after year, has led to a decline in production quality and an increase in pests and diseases. This cultivation practice negatively affects the sustainable production of crops (Wu and Zhou \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Research indicates that the causes of continuous cropping obstacles can be divided into three main categories: deterioration of soil physicochemical properties, and root exudates coupled with the decomposition of root litters (Uehling, Entler et al. 2019). Among these, the accumulation of toxic substances and the imbalance of soil microbial communities are significant contributing factors.Following continuous cropping, root litters persist in the soil. While their decomposition can release nutrients, enhance levels of soil organic carbon, phosphorus, and potassium, and thereby improve soil physicochemical properties,the degradation process is in fact slow (Fu, Chen et al. 2021). In the short term, microbial activity often triggers a \"nitrogen competition\" effect, leading to nitrogen immobilization (Chen, Liu et al. 2014). Furthermore, the accumulated litters attract pathogen colonization and reduce populations of beneficial microorganisms, disrupting the balance of the soil microbial community and contributing to disease outbreaks (Wu, Jiao et al. 2017). For instance, common soil-borne diseases in continuous tomato cropping, such as Fusarium wilt and bacterial wilt, are primarily caused by the buildup of \u003cem\u003eFusarium oxysporum\u003c/em\u003e and \u003cem\u003eRalstonia solanacearum\u003c/em\u003e in the soil (Basco, Bisen et al. 2017, Yin, Zhang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, During decomposition, root litters also release phenolic allelochemicals such as coumaric acid and ferulic acid, which reduce soil pH and organic matter content, thus altering the soil's physicochemical properties (Wang, Yang et al. 2024). Although returning crop litters to the field offers certain agronomic benefits, these are frequently outweighed by the severe drawbacks induced by continuous cropping obstacles, which are detrimental to long-term sustainability. Therefore, accelerating the degradation of root litters represents a crucial measure for mitigating these obstacles.\u003c/p\u003e \u003cp\u003eMicroorganisms serve as decomposers in ecosystems and play a vital role in maintaining ecological balance. Due to their environmentally friendly and non-toxic degradation processes, microbial inoculants have been widely adopted (Sruthy, Shukla et al. 2023). Numerous studies have demonstrated the ability of microorganisms to degrade root litters and secondary metabolites. Many saprophytic fungi and \u003cem\u003eStreptomyces\u003c/em\u003e species can secrete various extracellular enzymes to break down soluble substances and lignin (Zhou, Fransen et al. 2025), Certain \u003cem\u003ePenicillium\u003c/em\u003e species utilize phenolic acids as a carbon source, directly decomposing toxic substances. Various microorganisms not only exhibit litter-degrading capabilities but also demonstrate additional beneficial traits. For instance, the combined application of Bacillus subtilis with humic acid and chitosan can suppress the abundance of pathogens in continuous tomato cropping soil, reduce phenolic acid content, and mitigate disease incidence (Qiu, Bao et al. 2025).Additionally, certain microorganisms can enhance plant disease resistance and exhibit growth-promoting properties. For instance, \u003cem\u003eStreptomyces\u003c/em\u003e sp. SK68 not only demonstrates multiple degrading enzyme activities but also increases tomato biomass and alleviates salt stress after inoculation (Damodharan, Palaniyandi et al. 2018).While many microorganisms show some effect in degrading root litters, a large proportion demonstrate low efficiency. Therefore, it is necessary to screen for highly efficient strains capable of degrading tomato root litters.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to isolate and purify microbial strains from tomato root litters degraded for 15 and 60 days. Microorganisms capable of degrading various components of the litters were screened, and strains with high degradation activity were selected based on enzyme assays in liquid enzyme‑production media.The degradation capabilities of the screened strains and their combinations were further evaluated through \u003cem\u003ein vitro\u003c/em\u003e root litter degradation assays. This work aims to provide effective strain resources for the subsequent development of bioagents and to offer a theoretical basis for alleviating tomato continuous cropping obstacles.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eRoot litter Degradation Experiment\u003c/h2\u003e \u003cp\u003eWe collected tomato root litters from continuous cropping soil. These litters were rinsed clean, dried until constant weight was achieved, and then crushed into small segments of 1\u0026ndash;2 cm. Continuous cropping tomato soil was collected in May 2018 from the experimental station of Northeast Agricultural University in Harbin, China (45\u0026deg;41\u0026prime;N, 126\u0026deg;37\u0026prime;E).1 g of tomato root litters was mixed with 100 g of continuous cropping soil in 250 ml culture flasks. The soil was used to cover and homogenize the litters, and soil moisture was maintained at 65% of the field water-holding capacity. Each sampling time point included three replicates, with four flasks per replicate, totaling 24 flasks. All flasks were incubated in the dark at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. Root litters were sampled at 15 days and 60 days for subsequent screening of tomato litter-degrading microorganisms.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStrain Isolation\u003c/h3\u003e\n\u003cp\u003eAfter rinsing off soil particles attached to the tomato root litters with sterile water and surface‑sterilizing, the root litters were fully ground using PBS standard buffer under aseptic conditions. The ground mixture was transferred into a sterilized conical flask to obtain a suspension, which was then serially diluted to concentration gradients of 10⁻\u0026sup1;, 10⁻\u0026sup2;, 10⁻\u0026sup3;, 10⁻⁴, 10⁻⁵, 10⁻⁶, and 10⁻⁷. A 200 \u0026micro;L aliquot of each dilution was spread onto Beef Extract Peptone Agar and Rose Bengal Agar for the isolation of bacteria and fungi, respectively. The plates were inverted and incubated in a constant-temperature incubator (Bacteria: 37\u0026deg;C for 24 hours; Fungi: 30\u0026deg;C). After colony formation, individual colonies were picked and streaked onto LB agar (for bacteria) or PDA agar (for fungi) to obtain pure isolates. All isolated bacteria and fungi were further purified on LB and PDA media, respectively. After culturing for 3\u0026ndash;5 days, the strains were stored at 4\u0026deg;C and subsequently inoculated onto various screening media for functional screening in the short term.\u003c/p\u003e\n\u003ch3\u003eScreening for Root litter-Degrading Microorganisms\u003c/h3\u003e\n\u003cp\u003eThe isolated strains were inoculated onto a soluble starch medium (composition: ammonium sulfate 0.5%, K₂HPO₄ 0.5%, soluble starch 0.5%, peptone 1.5%, beef extract 0.3%, agar 0.8%, distilled water, pH 7.0). After inoculation, the plates were inverted and incubated in a constant-temperature incubator (bacteria: 37\u0026deg;C for 24 hours; fungi: 30\u0026deg;C for 2 days). Iodine solution was then dropped onto the developed colonies, and single colonies surrounded by starch hydrolysis halos were selected. Strains forming larger clear halos were chosen for secondary screening (Ogbonnaya and Odiase \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMilk medium plates (composition: beef extract 0.3%, peptone 1%, NaCl 0.5%, agar 2%, pH 7.4\u0026ndash;7.6,supplemented with 1.5% autoclaved skim milk) were used for screening protein‑degrading strains. Inoculation was performed as described above (Ma, Li et al. 2024)(bacteria: 37\u0026deg;C for 24 h; fungi: 30\u0026deg;C for 3 days). Strains capable of forming clear zones were selected, and those producing larger halos were subjected to secondary screening.\u003c/p\u003e \u003cp\u003ePectin primary screening medium (composition: K₂HPO₄ 0.1%, MgSO₄ 0.05%, NaNO₃ 0.3%, FeSO₄ 0.001%, pectin 0.2%, agar 1.5%, pH 7.0) was used to screen for pectin‑degrading strains. After incubation, Congo Red solution (0.2%, w/v) was added to the plates and allowed to stain for 2 h. The dye solution was then discarded, and the plates were washed with NaCl (1 M) for destaining (KC, Upadhyaya et al. 2020). Strains forming larger clear zones were similarly selected for secondary screening.\u003c/p\u003e \u003cp\u003eAgar CMC‑Na medium (composition: CMC‑Na 1.5%, NH₄NO₃ 0.1%, yeast extract 0.1%, MgSO₄\u0026middot;7H₂O 0.5%, K₂HPO₄ 0.1%, agar 1.5%) was used for screening cellulose‑degrading strains (Wei, Pan et al. 2004). After incubation, Congo Red solution (0.1%, w/v) was applied for 30 min. The dye was discarded, and the plates were destained with NaCl (1 M). Strains producing larger clear zones were chosen for secondary screening.\u003c/p\u003e \u003cp\u003eGU-PDA medium (PDA supplemented with 0.1% guaiacol) and Azure B medium (composition: yeast extract 1%, glucose 1%, Azure B 0.01%, agar 2%) were used for screening lignin-degrading strains. Incubation was carried out under the following conditions: bacteria at 37\u0026deg;C for 2 days; fungi at 28\u0026deg;C for 7 days. Changes in colony color and morphology were observed. Strains producing brown colored halos or clear degradation zones were selected for secondary screening, respectively (Bibi and Bhatti).\u003c/p\u003e\n\u003ch3\u003eRescreening of Strains\u003c/h3\u003e\n\u003cp\u003eThe selected bacterial and fungal strains were inoculated into LB liquid medium and PDB medium, respectively, and cultured with shaking at 30\u0026deg;C and 170 rpm. When the bacterial OD600 or fungal spore concentration reached approximately 10⁷ CFU/mL, the cultures were inoculated at 5% (v/v) inoculum size into liquid enzyme‑production medium.Each treatment was performed in triplicate and incubated with shaking at a constant 30\u0026deg;C. Samples were taken on days 2, 4, 6, and 8. After collection, the samples were transferred to 50 mL centrifuge tubes and centrifuged at 10,000 rpm for 10 min.The supernatant (the crude enzyme extract) was collected for subsequent enzyme activity assays. Crude enzyme extracts for starch, protein, pectin, and cellulose degradation were prepared using the method described above.\u003c/p\u003e \u003cp\u003e For lignin-degrading enzyme production, 0.5 g of root litters was placed into a 50 mL conical flask, followed by the addition of 10 mL of HNHC liquid medium (composition: glucose 2%, ammonium tartrate 0.2%, K₂HPO₄ 0.1%, MgSO₄\u0026middot;7H₂O 0.05%, CuSO₄\u0026middot;5H₂O 0.001%, ZnSO₄\u0026middot;7H₂O 0.0005%, MnSO₄ 0.0005%, pH 6.0). The flasks were autoclaved(Baldrian and Snajdr \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). A 5 mm mycelial plug taken from the edge of a seven‑day‑old fungal colony grown on a plate was inoculated into each flask. For bacterial strains, 1 mL of a bacterial suspension at 10⁷ CFU/mL was added to each flask. Sampling was performed on days 7, 9, 11, 13, and 15. At each sampling point, 10 mL of sterile water was added to each flask, followed by homogenization at 4\u0026deg;C and 150 rpm for 2 h. The mixture was then transferred to 50 mL centrifuge tubes and centrifuged to obtain the crude lignin-degrading enzyme solution.\u003c/p\u003e \u003cp\u003eAmylase activity was determined using the DNS method. The reaction mixture was heated in a boiling water bath for 10 min and then cooled (Mishra and Behera \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The optical density of each sample and the reaction mixture was measured at 575 nm using a microplate reader. One unit of enzyme activity was defined as the amount of enzyme that released 1 \u0026micro;mol of glucose per minute under the assay conditions.\u003c/p\u003e \u003cp\u003eProtease activity was measured by the Folin-phenol method (Zhao, Wang et al. 2016). The optical density of the reaction mixture was measured at 680 nm using a microplate reader. One unit of enzyme activity was defined as the amount of enzyme required to produce 1 \u0026micro;mol of tyrosine per minute.\u003c/p\u003e \u003cp\u003ePectinase activity was also assayed using the DNS method. A 1% pectin solution was prepared in 0.2 M sodium acetate buffer (pH 5.5). Then, 0.2 mL of crude enzyme extract was mixed with 0.8 mL of the substrate and incubated at 50\u0026deg;C for 1 h. The reaction was terminated by adding 1.5 mL of DNS reagent, followed by heating in a boiling water bath for 10 min. The volume was adjusted to 9 mL, and absorbance was measured at 540 nm using a microplate reader. One unit of enzyme activity was defined as the amount of enzyme that released 1 \u0026micro;mol of reducing sugar (galacturonic acid equivalent) per minute.\u003c/p\u003e \u003cp\u003eCellulase activity was determined by the DNS method. A 1% sodium carboxymethyl cellulose solution was prepared in 0.2 M sodium acetate buffer (pH 5.0). Then, 0.1 mL of crude enzyme extract was mixed with 1.9 mL of the substrate and incubated at 50\u0026deg;C for 20 min. The reaction was terminated by adding 1.5 mL of DNS solution, followed by heating in a boiling water bath for 10 min. After cooling, the absorbance was measured at 520 nm using a microplate reader. One unit of enzyme activity was defined as the amount of enzyme required to produce 1 \u0026micro;g of glucose per minute.\u003c/p\u003e \u003cp\u003eLignin-degrading enzyme activities were determined separately for two enzymes: laccase was assayed using the ABTS method, Peroxidase activity was determined by the veratryl alcohol method. (Li, Zhu et al. 2014).\u003c/p\u003e\n\u003ch3\u003eStrain Identification\u003c/h3\u003e\n\u003cp\u003eGenomic DNA of bacterial and fungal strains was extracted using bacterial and fungal genomic DNA extraction kits, respectively. For fungi, the ITS region of the rRNA gene was amplified using the primers ITS1/ITS4 (Henry, Iwen et al. 2000). For bacteria, the 16S rRNA gene sequence was amplified using the primers 27F/1492R. All amplified products were sequenced. The obtained ITS and 16S rRNA gene sequences were compared with those of other strains in the GenBank database using the NCBI BLASTn tool. Phylogenetic trees were constructed using the neighbor-joining method in MEGA 11.0 software. The stability of the tree clades was assessed with bootstrap values based on 1000 replicates.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of Strain Compatibility and Degradation Efficiency\u003c/h2\u003e \u003cp\u003eCompatibility tests were conducted between highly efficient strains capable of degrading different components of root litters. The compatibility between fungal strains, as well as between fungal and bacterial strains, was assessed using the dual‑culture plate confrontation method (Santiago, Yagi et al. 2017). The compatibility between bacterial strains was determined using the cross-streak method. Based on these results, compatible strains were selected to establish microbial consortia designated as Z1, Z2, Z3.\u003c/p\u003e \u003cp\u003eTo determine the degradation efficiency of individual strains and consortia, 2 g of root litters were weighed and placed into 100 mL conical flasks. Each flask was supplemented with 20 mL of inorganic salt medium to submerge the litters. After sealing with sealing film and autoclaving, 2 mL of a highly efficient tomato root litter‑degrading bacterial suspension (concentration: 10⁷ CFU/mL) was inoculated into each flask. Each treatment was performed in triplicate. Individual strains and the constructed consortia served as experimental groups. A control group was set up by adding the same volume of magnesium sulfate solution without bacterial inoculation. All flasks were incubated in the dark at 30\u0026deg;C. Samples were taken on day 15(Li, Wang et al. 2011, Yang, Wang et al. 2017). The root litters were rinsed with sterile water, oven‑dried at 80\u0026deg;C, and weighed. The degradation rate of tomato root litters was calculated using the weight‑loss method: Degradation rate (%) = [(m₀ \u0026ndash; m) / m₀] \u0026times; 100%, where m₀ is the initial weight of the root litters and m is the weight of the root litters after rinsing and drying on day 15 following inoculation (Zhang, Wang et al. 2022).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experimental data were analyzed using one-way analysis of variance (one‑way ANOVA), followed by multiple comparisons with the Tukey HSD test. The significance level was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All bar charts and line graphs were generated using Origin Pro 8.5.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eScreening of Root litter-Degrading Microorganisms\u003c/h2\u003e \u003cp\u003ePrimary screening results showed that 477 bacterial and 417 fungal with degradation ability were obtained from the 15‑day degraded root litters, while 713 bacterial and 408 fungal were isolated from the 60‑day degraded litters. Larger degradation halos indicate stronger degradation ability. Strains exhibiting larger halos were therefore selected for rescreening.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRescreening of Strains\u003c/h2\u003e \u003cp\u003eBased on the primary screening results, 16 strains exhibiting degrading enzyme activities were selected. Among them, the fungal strain \u003cem\u003eSimplicillium\u003c/em\u003e sp. bb60‑73 showed the highest amylase activity, reaching 6.8 U/mL on day 2 (Figure.1a). The highest protease activity was observed in the bacterial strain \u003cem\u003ePaenibacillus\u003c/em\u003e sp. ab60‑72, which remained stable between 4.0 and 4.5 U/mL (Figure.1b). For pectinase, the fungal strain \u003cem\u003eAlbifimbria verrucaria\u003c/em\u003e bc15-20 exhibited the highest activity, peaking at 4.4 U/mL on day 2 (Figure.1c). The bacterial strain \u003cem\u003ePaenibacillus\u003c/em\u003e sp. bc60‑90 displayed the highest cellulase activity, reaching 1.55 U/mL on day 8 (Figure.1d). Regarding lignin‑degrading enzymes, the highest laccase activity was recorded for the fungal strain \u003cem\u003eTrametes\u003c/em\u003e sp. ab60‑59 (2.2 U/mL) (Figure.1e), and the highest peroxidase activity for the fungal strain \u003cem\u003ePenicillium\u003c/em\u003e sp. ca60‑13 (4.56 U/mL) (Figure.1f), both peaking on day 15.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of Degrading Strains\u003c/h2\u003e \u003cp\u003eThe 16S rRNA and ITS1 gene sequences of the 16 selected degrading strains were obtained for homology analysis. Sequences with similarity above 99% were selected to construct a phylogenetic tree using MEGA 11.0. Strains ca60‑52(PX970998A) and ba60‑13(PX970997A) were preliminarily identified as bacteria belonging to the genus \u003cem\u003eMicrobacterium\u003c/em\u003e; strain bb60‑111(PX964323A) was identified as \u003cem\u003eCellulosimicrobium funkei\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). strains bb60‑72(PX964323A) and bc60‑90(PX964330A) were assigned to the genus \u003cem\u003ePaenibacillus\u003c/em\u003e. Among the fungal strains, bc60‑61(PX963975A), aa60‑12(PX963889A), and ac60‑80(PX963974A) were identified as members of the genus \u003cem\u003ePenicillium\u003c/em\u003e; ca60‑51(PX963842A) and bc60‑63(PX964219A) were identified as \u003cem\u003eAspergillus\u003c/em\u003e sp; ab60‑73(PX963963A) was identified as \u003cem\u003eSimplicillium\u003c/em\u003e sp.; bb60‑2(PX963973A) was identified as \u003cem\u003eSarocladium strictum\u003c/em\u003e; cb60‑55(PX964257A) and bc15-20(PX975913) was identified as \u003cem\u003eParamyrothecium roridum\u003c/em\u003e; ab60‑59(PX963890A) was preliminarily identified as a fungus of the genus \u003cem\u003eTrametes\u003c/em\u003e; and ca15‑65(PX964223A) was identified as a \u003cem\u003eFusarium\u003c/em\u003e sp (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The 16S rDNA and ITS1 gene sequences of the strains were submitted to GenBank to obtain the sequence accession numbers.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of Strain Compatibility and Degradation Rate\u003c/h2\u003e \u003cp\u003ePairwise compatibility tests among the 16 degrading strains revealed that fungal strain aa60‑12 was incompatible with fungal strains bc15-20 and ab60‑73, while all other strain combinations were compatible, Based on the compatibility results, strains were selected from the compatible isolates and used to construct microbial consortia. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The 16 screened degrading strains were subjected to an \u003cem\u003ein vitro\u003c/em\u003e root litter degradation test. On day 15, fungal strain bc60‑61 showed the highest degradation rate (39.56%), while bacterial strain bb60‑72 exhibited the lowest (11.33%). With the exception of fungal strain aa60‑12, all other fungal strains demonstrated greater degradation efficiency than the bacterial strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In contrast, compatible consortia achieved the following degradation rates: Z1 (bc15-20 and ab60‑73) 26.56%, Z2 (bc60-63, bb60‑2, bb60-111, and aa60-12) 33.49%, and Z3 (bc60‑63, bb60‑2, bb60-111, and ba60-13) 34.18%.Among these, the degradation rate of consortium Z1 was comparable to that of its constituent single strains. Consortia Z2 and Z3 exhibited significantly higher degradation rates than any individual strain within the respective groups, demonstrating a pronounced synergistic effect on tomato root litter degradation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, microorganisms were isolated and purified from tomato root litters degraded for 15 and 60 days using the dilution plating method. It was observed that as the degradation period extended, the number of bacterial colonies decreased, while the number of fungal colonies increased. This phenomenon may be attributed to the faster growth rate of bacteria during the early stages of decomposition, allowing them to preferentially utilize available nutrients (Rousk, Brookes et al. 2010).In contrast, fungi, despite their slower initial growth, are capable of secreting a wider array of enzymes that degrade recalcitrant substances in the later phases of decomposition (Lorenz and Lal \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Poll, Marhan et al. 2008). These findings are consistent with the results obtained in our experiments.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e root litter degradation assay revealed that fungi overall exhibited stronger degradation capabilities compared to bacteria. Among them, the \u003cem\u003ePenicillium\u003c/em\u003e strain bc60‑61 showed the highest degradation ability. Fungi from the genera \u003cem\u003eSimplicillium\u003c/em\u003e and \u003cem\u003eAspergillus\u003c/em\u003e were not only the most abundant but also demonstrated strong degradation performance (Hahn Schneider, Goncalves et al. 2016). \u003cem\u003eBasidiomycete\u003c/em\u003e strains such as bc15-20 and cb60‑55 also possessed relatively high degradation abilities for cellulose and lignin. This may be attributed to the more comprehensive repertoire of enzymes secreted by fungi, enabling them to effectively degrade lignin\u0026mdash;the primary component of root litters.The fungi isolated in this study predominantly belonged to Ascomycota, which is consistent with reports indicating that Ascomycota fungi play a dominant role during the early to middle stages of litter degradation (Deacon, Pryce-Miller et al. 2006). The \u003cem\u003ePenicillium\u003c/em\u003e and \u003cem\u003eAspergillus\u003c/em\u003e strains screened here exhibited strong root litter degradation ability, with bc60‑61 performing the best. This high efficiency may be attributed to the secretion of various hydrolytic enzymes and antibiotics by these fungal genera, which can suppress competing microorganisms, including pathogens, thereby facilitating their dominance in the decomposition process (Hiscox and Boddy \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Some of the screened strains belonged to the genus \u003cem\u003eFusarium\u003c/em\u003e, which is frequently associated with tomato wilt diseases. However, whether strain ca15‑65 is pathogenic remains unclear and requires further experimental verification.Among the bacterial isolates, strains from the phylum Actinobacteria demonstrated relatively strong degradation capabilities. Strains bb60‑111 and ca60‑52, all belonging to Actinobacteria, exhibited higher degradation activity than those from the phylum Firmicutes.Notably, strain ba60‑13 also showed the ability to degrade cellulose and was the most efficient bacterial strain in degrading root litters. This can be attributed to the capacity of Actinobacteria to secrete a variety of hydrolytic enzymes, endowing them with strong \u003cem\u003ein vitro\u003c/em\u003e degradation potential for plant litters (Bao, Dolfing et al. 2021).\u003c/p\u003e \u003cp\u003eStudies have shown that microorganisms can interact and communicate with each other, and the construction of microbial communities can enhance the degradation ability of root litters (Sruthy, Shukla et al. 2023, Singh, Abiraami et al. 2025). In this study, compatibility tests were conducted among the 16 screened strains. The compatible consortium Z1, consisting of bc15-20 and ab60‑73, did not exhibit a significant improvement in degradation rate, suggesting that these two strains neither strongly synergize nor inhibit each other under the tested conditions. Consortia Z2 and Z3 demonstrated significantly enhanced degradation capabilities compared to their individual constituent strains, indicating that compatible multi‑species combinations improve degradation efficiency. Fungi can efficiently decompose root litters, and the intermediate products are fully utilized by bacteria (Zhang, Wen et al. 2025), creating a synergistic relationship. Moreover, fungi can secrete compounds that promote bacterial growth (Uehling, Entler et al. 2019). Increased microbial diversity also strengthens the stability of the community, and the various metabolites produced can directly or indirectly suppress pathogen spread and proliferation (Singh, Jiang et al. 2025). Similarly, combinations of four Firmicutes bacteria enhanced plant immune responses and improved tomato resistance compared to single or paired bacterial treatments (Lee, Kong et al. 2021).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, using tomato root litters degraded for 15 and 60 days as materials, 11 fungal strains and 5 bacterial strains capable of degrading root litter components were isolated. The degradation ability of fungi was generally higher than that of bacteria, with fungi from genera such as \u003cem\u003ePenicillium\u003c/em\u003e, \u003cem\u003eAspergillus\u003c/em\u003e, \u003cem\u003ePaecilomyces\u003c/em\u003e, and \u003cem\u003eFusarium\u003c/em\u003e showing a more pronounced advantage. Strain compatibility assays revealed that certain compatible consortia enhanced the degradation efficiency of root litters. This work lays a foundation for subsequently constructing microbial consortia for tomato root litter degradation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Natural Science Foundation of China [32573013, 32402556].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo potential conflict of interest was reported by the author(s).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBaldrian P, Snajdr J (2006) Production of ligninolytic enzymes by litter-decomposing fungi and their ability to decolorize synthetic dyes. 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Here, we isolated bacterial and fungal strains capable of degrading tomato root litter and construct efficient cross-kingdom microbial consortium. Through \u003cem\u003ein vitro\u003c/em\u003e screening, we obtained 16 strains, including five bacterial strains and eleven fungal strains that can degrade tomato root litter. The degradation abilities of the fungal strains were generally higher than that of the bacterial strains. Then, we successfully contrasted two cross-kingdom microbial consortiums Z2 (containing \u003cem\u003eAspergillus\u003c/em\u003e sp., \u003cem\u003eSarocladium strictum\u003c/em\u003e, \u003cem\u003eCellulosimicrobium funkei\u003c/em\u003e, and \u003cem\u003ePenicillium\u003c/em\u003e sp.) and consortium Z3 (containing \u003cem\u003eAspergillus\u003c/em\u003e sp., \u003cem\u003eSarocladium strictum\u003c/em\u003e, \u003cem\u003eCellulosimicrobium funkei\u003c/em\u003e, and \u003cem\u003eMicrobacterium\u003c/em\u003e sp.) that could efficiently degrade tomato root litter. The microbial strains in each consortium were compatible to each other. These two microbial consortia showed higher degrading efficiency than any single strain in each consortium. The results indicate that fungi play a dominant role in the degradation of tomato root litters, and certain compatible strain consortia can enhance the degradation capacity.\u003c/p\u003e","manuscriptTitle":"Cross-kingdom microbial Consortiums efficiently Degradate Tomato Root litter","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-24 00:24:53","doi":"10.21203/rs.3.rs-9101939/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-04-16T08:53:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-14T14:05:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-14T04:31:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Current Microbiology","date":"2026-03-12T07:57:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":false,"email":"","identity":"current-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Current Microbiology","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9a515b31-127c-4c11-bebb-9f9d9fe280d5","owner":[],"postedDate":"April 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-24T00:24:53+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-24 00:24:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9101939","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9101939","identity":"rs-9101939","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}