Life cycle of the Root-Knot Nematode Meloidogyne javanica in papaya and histological analyses of root infection and gall development.

Life cycle of the Root-Knot Nematode Meloidogyne javanica in papaya and histological analyses of root infection and gall development. | 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 Life cycle of the Root-Knot Nematode Meloidogyne javanica in papaya and histological analyses of root infection and gall development. Kouroubi R.L. COULIBALY, Bouma THIO, Myriam COLLIN, Kadidia KOITA, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5377678/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Papaya ( Carica papaya L.) is susceptible to attacks by root-knot nematodes (RKN), which lead to significant production losses. Understanding the life cycle of RKN in papaya is essential for developing effective control strategies and screening for natural resistance in papaya cultivars. In this study, the development of the RKN Meloidogyne javanica was assessed in the susceptible papaya variety Solo8 over a period of 35 days, using microscopic observation of inoculated roots stained with fuchsin. By 9 days after inoculation (DAI), second-stage juveniles (J2s) were observed migrating through the root cortex and feeding within the vascular cylinder. At 16 DAI, galls containing enlarged J2s had formed within the stele, where feeding sites were established. Young females began appearing within the galls at 26 DAI, and mature pear-shaped females with egg masses were present by 35 DAI. Remarkably, some egg masses were deposited within the cortex, where a new generation of J2s hatched inside the root. Histological cross-sections of galls revealed that feeding sites initiated around the nematode head within the stele by 9 DAI, with four to eight multinucleated giant cells (GCs) present at these sites. These GCs, located within xylem and protophloem elements, were progressively encased by additional cell layers from adjacent vascular and cortical tissues as the nematode developed within the gall at 16, 26, and 35 DAI. The GCs reached maximum size by 16 DAI. Cytoplasmic analysis showed that GCs were rich in proteins, as evidenced by Naphtol Blue Black staining, and that their cell walls were strongly stained for polysaccharides using PAS. This study offers comprehensive histological insights into nematode development within papaya roots, underscoring that screening papaya genotypes for RKN resistance should consider egg mass production within the root tissue. Carica papaya gall giant cell histology microscopy nematology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Papaya ( Carica papaya L.) is a tropical fruit crop grown in various tropical and subtropical regions around the world. Its rich nutritional value, unique flavor, and versatility have made it a popular choice among consumers worldwide. However, the successful cultivation of papaya is often hampered by various biotic and abiotic factors, with plant-parasitic nematodes emerging as one of the primary challenges. Nematodes are microscopic worms that inhabit the soil and attack the roots of plants. They inflict damage by feeding on the roots, causing root galling, stunting, and reduced nutrient uptake. The most common nematode species affecting papaya include the reniform nematode Rotylenchulus reniformis and root-knot nematodes (RKN) of the genus Meloidogyne (Goëldi 1892). A recent survey in Burkina Faso showed that RKNs ( Meloidogyne incognita and Meloidogyne javanica ) are commonly found in several regions of papaya cultivation (KRL Coulibaly and B Thio, pers. com.) These nematodes can drastically reduce fruit yield, leading to economic losses for farmers and impeding the growth of the papaya industry. Managing plant-parasitic nematodes in papaya cultivation necessitates an integrated approach combining cultural, biological, and chemical methods. Use of resistant/tolerant cultivars is a promising way to reduce damages caused by RKN (Kaloshian and Teixeira 2019 ). A breeding program is currently developed at INERA, Burkina to produce papaya varieties that can afford nematode infection. However, assessing nematode pathogenicity, and evaluating the efficacy of resistant/tolerant cultivars requires good knowledge about the life cycle of the nematodes into the roots. RKN are sedentary nematodes that settle in roots and feed on specialized cells during most stages of their life cycle that comprises five developmental stages separated by 4 molts (Tryantaphyllou and Hirschmann 1960; Eisenback 1985 ; Escobar et al. 2015 ). The infective stage of Meloidogyne is the second-stage juvenile (J2) that hatch from eggs in soil and is specifically attracted by root exudates towards host plant roots. J2s can directly penetrate the root apex and migrate through the cortex, moving between cells, to reach the vascular tissues. The nematode use a combination of mechanical piercing through its stylet and cell wall softening by producing specific carbohydrates degrading enzymes (CAZymes) to enter and migrate into roots (Wieczorek 2015 ). Upon reaching the vascular cylinder, J2s settle and start feeding on certain parenchyma cells. Salivary secretions produced by the nematode induce the transformation of the parenchyma cells into hypertrophied and multinucleated "giant cells" (GC) encaged within a network of de novo formed xylem and phloem cells which serve as active feeding sites for the nematode (Bartlem et al. 2013 ). In addition, GC formation is accompanied by proliferation of adjacent cells resulting in visible root deformation known as gall, disrupting the normal root architecture and growth (Kyndt et al. 2013 ). Sedentary nematodes within galls feed on giant cells (GC) to progress through their larval development from the third-stage (J3) and fourth-stage (J4) to adulthood. Sexual differentiation into males or females may occur based on environmental conditions. Under unfavorable conditions, males may develop; however, in most Meloidogyne species, females that reproduce parthenogenetically are typically produced. They then lay eggs in a gelatinous matrix outside the root, with exception of Meloidogyne graminicola that lay eggs inside the root cortex of rice ( Oryza sativa L.) (Fernandez et al. 2015 ). The larvae that hatch from these eggs can reinfect the same root or migrate into the soil to infect other plants. The ability of Meloidogyne to manipulate plant cell metabolism to create their feeding site, without activating the innate immune system of their host is a key factor in their parasitic success (Rutter et al. 2022 ). In resistant plants, the RKN life cycle may be stopped at pre-infection stages preventing juveniles attraction and penetration or in subsequent post-infection stages impeding GC formation or development until female production (Goverse and Smant 2014 ; Goode and Mitchum 2022 ). The histology of RKN – host interaction has been described in several plant species highlighting the specific features of GC and gall development in susceptible and resistant plants (for review Escobar et al. 2015 ). Host immune reactions include different mechanisms underlying basal and specific nematode resistance, including callose deposition and typical hypersensitive responses (HR) blocking the nematode in an early stage (Albuquerque et al. 2010 ) or the feeding structure collapses because of nuclear and cytoplasmic degeneration (Petitot et al. 2017 ). RKN development in papaya roots was never examined. In the field, the variety Solo is susceptible to RKN. We present, for the first time, an analysis of RKN development in papaya roots and the histological alterations induced in host tissues. This investigation utilized a combination of light microscopy techniques, including the observation of fuchsin-stained nematodes and a detailed histological analysis of root galls and GC induced by the nematode. Material and Methods Culture and inoculation of nematodes A population of Meloidogybe javanica was isolated from an egg mass taken from papaya roots of the FBPA1 variety in the INERA Farako Bâ research station, (4°20'W; 11°06'N) near the town of Bobo-Dioulasso, Burkina Faso. The identification of the species M. javanica was confirmed following molecular characterization using the specific SCAR marker developed by Zijlstra et al. ( 2000 ). The multiplication of the nematode was carried out on tomato plants ( Solanum lycopersicum L. cv. Naine) in a culture chamber (26°C/24°C day/night temperature, under a 14-h day/10-h night light regime with a Photosynthetic Photon Flux Density (PPFD) of 210 µmol/s.m2) with 78% relative humidity (RH) at Institut de Recherche pour le Développement (IRD), Montpellier, France. Three months after inoculation, the nematode eggs were recovered by immersing the roots in 0.5% sodium hypochlorite for 3 minutes before mixing them in a blender for 5 times during 1s. Eggs were collected onto a 25 µm mesh and were hatched at 28°C. Second stage juveniles (J2) collected after a maximum of 72 h were counted and used as inoculum. Cultivation of papaya trees and inoculation of M. javanica Papaya seeds of the cultivated Solo variety (n°8 Technisem lot n° 31072-0820-98) were germinated on sand wet with Hoagland's solution (diluted 4 times) (KNO3 5 mM; KH2PO4 1 mM; Ca(NO3)2 5 mM; MgSO4 2 mM; 25 mg iron (Fe-EDDHA); trace elements) and maintained in a culture chamber (same conditions as for nematodes). After germination (3–4 weeks long), the papaya seedlings were transferred to a 26 mm-diameter PVC tube (Fig. 1 ) filled with SAP (sand and water-absorbent synthetic polymer) (Reversat et al. 1999) and watered 3 times per week with Hoagland's solution (diluted 4 times). Papaya plantlets were inoculated after 3–4 weeks of growth by carefully opening the SAP cylinder contained into the PVC tube and bringing an aqueous suspension containing the J2 of M. javanica all along the exposed root system using a micropipette. At this stage, papaya root systems were ca. 15-20-cm long and several lateral roots arised from the main root. Two inoculation assays were conducted (on June 15 and June 20, 2022). In the first assay, papaya seedlings were inoculated with 400 M. javanica J2, followed by the second assay in which seedlings were each inoculated with 2000 J2. Monitoring the infectious process by fuschin staining Papaya roots infested with M. javanica were collected at 9, 16, 26 and 35 days after inoculation (DAI), stained by immersing during 1 min in boiling acid fuschin, rinsed with osmosis water and placed in acidified glycerol according to the method of Byrd et al. ( 1983 ). Roots were first observed under a binocular loop, and the infested parts were dissected and mounted in glycerol between slide and coverslip. Nematode developmental stages (J2, feeding or not, young females, mature females and eggs) were assessed under the Leica DM1000 microscope (40x and 100x magnification) following determination of Tryantaphyllou and Hirschmann (1960). However, we could not determine the sexual stage of feeding J2 nor the J3 and J4 stages with precision. Typical developmental stages were recorded and photographed to take nematode measures (length and width). The Zeiss Axio Imager Z2 microscope equipped with an Axiocam 506 camera was used to take the photos. Histological analysis of the infection of papaya roots Some root tip fragments of 0.5 to 1 cm or ca. 2–4 visible galls on the root system were randomly taken from plants used for fuchsin staining at 9, 16, 26 and 35 DAI. Samples from each plant were processed separately during all stages, embedded in separate blocks and histological data were recorded from sections obtained from each block at each time point. Root and gall samples were immediately immersed in a fixative (2% paraformaldehyde, 1% glutaraldehyde and 1% caffeine), infiltrated under vacuum for 15 minutes and incubated at 4°C for 3 days. Then, the samples were dehydrated in 30% and 50% ethanol for 1 h each and then kept in 70% ethanol. The dehydration of the samples continued in ethanol at increasing concentration (80%, 90%, 95% and 100%) for 1 hour at room temperature before storage in 100% ethanol overnight at 4°C. The fixed root and gall samples were embedded in resin (epoxy Technovit 7100, Kulzer Friedrichsdorf, Germany) and blocks were sectioned using the Epredia™ HM 355S microtome (Fischer Thermo Scientific, France). Sections (10 µm width) were mounted in slides (3 sections per slide) and examined without staining to detect large cells typical of feeding sites. Sections with feeding sites were initially stained with 0.05% toluidine blue in 0.1 M sodium phosphate buffer (PBS), pH 5.5 (3 min at room temperature) for cytological observations. To detect soluble proteins in cells, sections were stained with Naphthol Blue Black (NBB). Polysaccharides were detected with PAS, for Periodic Acid 2% for 5 minutes followed by Schiff 1% in PBS for 15 minutes in the dark. Lignins and cellulose were detected with Fasga (diluted 1/7 in PBS for 3 h), and nucleic acids with Dapi (1/100 in PBS for 5 minutes). After staining, the sections were dried and mounted between slide and coverslip for microscopic observations. Images were taken using the Zeiss Axio Imager Z2 microscope and ZEN 3.6 blue edition software. Results Nematode cycle in papaya roots The developmental cycle of M. javanica was assessed by staining the nematodes in roots with acid fuchsin at 9, 16, 26, and 35 DAI. This time course was arbitrarily determined based on preliminary trials with a small number of plants. Two assays were conducted with four plants inoculated with same initial densities (ID) of juveniles of M. javanica analysed at each time point, and nematodes were observed and counted in roots. Depending on the number of nematodes inoculated on each root system, an average (mean ± SD; n = 12 ) of 65 ± 28 (ID = 400 J2) to 177 ± 104 (ID = 2000 J2) juveniles successfully penetrated and established in papaya roots. On average, 32 (ID = 400 J2) to 62 (ID = 2000 J2) galls formed on each root system. Typical nematode developmental stages observed are presented in Fig. 2 . At day 9, papaya roots exhibited slight swellings, with barely visible galls, especially at the root tips. Nematodes were distributed along the root system, from tip to 10 cm up. Inside roots, larvae were mostly found near or inside the stele. M. javanica larvae remained vermiform typical of the J2 infective stage either filiform (15 ± 2 µm in width; 378 ± 28 µm in length, n = 19 ) (Fig. 2 a), or showing a slight increase in width (25 ± 6 µm, n = 19 ) (Fig. 2 b), suggesting that feeding on plant has started. At day 16, visible galls had formed and were scattered throughout the root system. Nematodes located within the galls were predominantly found in the cortex, oriented with their heads toward the vascular cylinder, and had increased in thickness without a corresponding increase in length, indicating active feeding on the plant. Some feeding J2 remained vermiform, similar in size to those observed at 9 DAI (Fig. 2 b), while others had developed a swollen, "sausage-like" shape (56 ± 8 µm in width, n = 10 ) (Fig. 2 c). Additionally, several nematodes exhibited a more saccate form with a spiked tail, suggesting they were shortly before the second molt transitioning into the third-stage juvenile (J3) (82 ± 17 µm in width; 381 ± 41 µm in length, n = 12 ) (Fig. 2 d). At day 26, larger galls developed throughout the root system. Three M. javanica developmental stages were observed, with nematodes either close to stages J3 as at day 16 (Fig. 2 d), or young females with rounded posterior end (Fig. 2 e) or pear-shaped mature females (Fig. 2 f). No male was observed. Female body had considerably increased in size. Young females measured 191 ± 59 µm large (posterior end) and 515 ± 90 µm long ( n = 14 ), and mature females reached 312 ± 53 large (posterior end) and 579 ± 75 long ( n = 17 ). Feeding sites with pink-stained GCs were observed near the female heads embedded in the root vascular cylinder. At day 35, most M. javanica observed in galls were mature females with a pyriform shape and an egg mass attached to their posterior end (Fig. 2 f). Some egg masses protruded outside the papaya root but the majority of them remained enclosed in the cortical root tissue. Female size was up to 446 ± 76 µm large (posterior end) and 723 ± 104 long ( n = 43 ). Newly hatched J2s of the next generation were also observed in vicinity of egg masses. Figure 3 summarizes the two experiments showing the percentage of nematodes at different developmental stages (infective J2, young females, mature females, and newly hatched J2) during the infection of papaya roots by M. javanica . In both experiments (1 and 2), J2 stages accounted for 100% of the population at 9 days DAI and over 99% at 16 DAI indicating that the initial penetration and establishment phases were synchronous across experiments. The majority of immature young females were observed starting at 26 DAI, making up more than 80% of the total population. By 35 DAI, only mature females with eggs were observed, alongside approximately 10–15% of newly hatched J2. These data show that the M. javanica cycle in papaya, from J2 penetration to the next generation J2 release, has a duration of approximately 35 DAI at 28°C. Histological analysis of the infection of papaya roots with M. javanica Root tips and galls were collected at different times after inoculation to highlight the histological structure of the feeding sites induced by M. javanica in papaya. Examination of longitudinal root sections and transverse gall sections stained with toluidine blue or NBB-PAS allowed observation of the morphology of GCs and their subcellular components, as well as of the surrounding cells at different stages post-infection (Fig. 4 ). Several sections of at least 20 feeding sites were observed at each time point. At day 9, histological blocks contained mostly swollen root tips and few galls. Longitudinal sections of roots showed well structured cell layers, including epidermis, cortical parenchyma, pericycle, endodermis and vascular parenchyma (Fig. 4 a,b). Several hypertrophied vascular parenchymatic cells characteristic of GCs are found associated to J2 inside the root vascular cylinder close to the meristeme or higher on the root (Fig. 4 a,b). Approximately 4 to 7 GCs per feeding site are visible on each root section plane. GC are multinucleated, with 4–10 nuclei with large dense nucleolus (deep-blue stained) on each cell section indicating that intense DNA replication was taking place. The cytoplasm of giant cells appears dense and contains 1–2 large vacuoles. A discrete proliferation of vascular parenchymatic cells adjacent to the CGs is observed. The nematodes visible on the sections had increased in size compared to the inoculated J2. Importantly, all the feeding sites observed were located inside the vascular cylinder, well delimited by and no feeding sites was found in the root cortex. At day 16, several feeding sites with swollen feeding nematodes are observed in all sectioned galls (Fig. 4 c, d). The CGs are housed in the vascular cylinder that conserved its integrity despite swelling. GCs had largely increased in size compared to day 9 (Fig. 5 ). They present thick walls and their dense, granular-looking cytoplasm contains several small vacuoles. Several nuclei with large dense nucleolus are well visible. Around 4 to 8 CGs are observed per feeding site and look embedded with newly formed xylem vessels and phloem elements. Xylem elements can be identified by the thick cells walls, differential toluidine blue or NBB staining, and banding patterns of lignified cell wall thickenings (Fig. 4 b, e, f). In contrast, the phloem appears as sieve elements that are frequently nucleated. Increased proliferation of adjacent cells into several (4–5) layers was observed from both sides of GCs in longitudinal sections (Fig. 4 d). The newly formed cells apparently originated from the pericycle in vascular cylinder as well as from the root cortex. The new vascular parenchymatic cells, probably phloem elements, lost their typical rectangular shape and presented irregular shapes encircling the giant-feeding cells. All nematodes retained their elongated form, likely representing J2s. Nematode head was embedded among CGs while their body lied inside the vascular parenchyma (Fig. 4 c). At day 26, several well-developed feeding sites were observed near the nematodes within the vascular cylinder, all visible in the same cross-sectional plane (Fig. 4 e, f). GCs with thickened walls, asymmetrical shapes, numerous protrusions, and dense cytoplasm containing few vacuoles were noted. On NBB-PAS-stained sections, the blue staining of the giant cell cytoplasm, along with the proliferation of pink-stained parenchymal cells, became more pronounced at this stage of development. Feeding sites appear well delimited in the stele that gained 4–5 additional surrounding cell layers. Some nematodes displayed a rounded shape characteristic of young females (Fig. 4 e), suggesting that they underwent molts (J2-J3-J4-young females). Sometimes, the induction of lateral roots originating from pericycle cells was also visible situated at the feeding site. At day 35, numerous feeding sites were observed within the vascular cylinder, accompanied by increased proliferation of adjacent cells (Fig. 5 a). The GCs were generally not visibly different from those formed 26 DAI. The majority of nematodes were mature egg-laying females with egg masses in a gelatinous matrix deposited either inside the cortex or outside the cortex. Females had their posterior body part, now of piriform shape, located in the cortex, and a long anterior ‘neck’ and head embedded in a group of GCs located in the vascular cylinder. The surface area of GCs was measured on the two to three largest GCs per feeding site, with 54 giant cell sections recorded at each time point (Fig. 6 ). On day 9, the maximum GC surface area measured in a section was 22,688 µm². A substantial increase in GC surface area was observed by 16 DAI, with the highest recorded value reaching 94,781 µm². No further increase in GC surface area was noted at subsequent time points. In comparison, the surface areas of vascular parenchymatic and cortical cells, measured in the same sections, showed no significant variation over time, with averages of 348 ± 59 µm² and 5,227 ± 1,243 µm², respectively (Table 1, supplemental data). GCs function as nutrient source tissues, supplying nematodes with soluble sugars and other essential molecules transported from the aerial parts of the plant. Sections were stained using a combination of NBB and PAS dyes. NBB stains proteins blue, while PAS highlights polysaccharides (glycogen, starch or cellulose) in pink. In all NBB-PAS-stained sections presented in Figs. 4 and 5 a, both the nematode and GC cytoplasm appeared uniformly blue, with the thickened walls of GCs and other root cell walls stained a deep pink. Nuclei in all cells presented a dark blue nucleolus, indicating a high protein content probably reflecting the ribosome biosynthesis activity. Additionally, DAPI dye characteristic of nucleic acids confirmed nuclei identification in GC (Fig. 5 d). When PAS alone was applied, polysaccharides were revealed in cell walls (cellulose) and GCs cytoplasm was stained as uniformly pale pink (Fig. 5 c), indicative of weak presence of glycogen or starch, but no starch grain were visible. To further investigate the gall tissue structure and cell wall components, some sections were stained using FASGA, a mixture of fuchsin, alcian blue, safranin, and glycerin, which stains cellulose (and pectin) blue-green and lignin red (Tolivia and Tolivia, 1987 ). FASGA stained all root cell walls blue-green, including the thickened GC walls, and did not reveal lignin deposition (Fig. 5 b). Red staining was only observed in certain deposits within xylem vessels (data not shown). The nematode itself did not stain with either FASGA dye. Discussion In this study, we assessed the key developmental stages of M. javanica in papaya (variety Solo) roots during 35 days. In addition, the histological examination of papaya roots during infection provided crucial insights into the dynamic interactions between the nematode and the host plant. The analysis highlights how the development of feeding sites, particularly GCs, progresses over time and the impact of this process on root tissue architecture. These findings are consistent with previous studies on RKN infecting other host plants (Perry et al. 2009 ) yet exhibiting some differences and contribute to a broader understanding of plant-nematode interactions. M. javanica cycle in papaya lasted 5 weeks in our culture chamber conditions (Fig. 3 ). By 35 DAI, the nematodes were all mature females, and egg masses were observed in galls. The large size of piriform females (up to 723 µm in length) and the presence of newly hatched J2s in the vicinity of egg masses confirmed the completion of the reproductive cycle. This observation mirrors findings in other studies that note the Meloidogyne life cycle typically culminates in the production of egg masses within 4–5 weeks under optimal conditions (Bird, 1959 ; Dropkin, 1963 ). M. javanica has a large host range and is commonly found in tropical areas (Evans and Perry, 2009 ). In rice ( O. sativa ), the M. javanica cycle has a similar duration with all females laying eggs at 28 DAI in the same experimental conditions (Grossi de Sá et al. 2019). Interestingly, several M. javanica egg masses were found inside the root cortex of papaya (Fig. 2 ). This feature is common in M. graminicola , a RKN species that attacks several graminaceous hosts including rice, but was never reported for M. javanica . M. javanica can attack rice grown in rainfed systems but produce eggs outside the root (Grossi de Sá et al. 2019). Egg-laying in the cortex has been thought to be an adaptation of M. graminicola to flooding conditions in irrigated rice fields. However, this is not the case for papaya cultivation and it is questioning whether it could be an adaptation to this particular plant species. Nematode females produce via vulval secretions some plant cell wall modifying proteins for the breakdown of root cells in order to adjust space for their body enlargement and likely to expulse their eggs at root surface (Vieira et al. 2011 ). It is therefore possible that in some strong papaya roots M. javanica females will not be able to produce enough degrading secretions to expulse their eggs. The presence of hidden M. javanica egg masses inside roots is an important data that breeders should take into account when screening papaya varieties for resistance to RKNs. Egg mass production on roots is frequently used as a parameter for plant resistance to nematodes that may be underestimated in the case of papaya – M. javanica interactions. The infection process of M. javanica in papaya roots followed a well-documented developmental timeline of RKNs, characterized by the formation of galls, the establishment of GCs in the stele, and the maturation of nematodes into egg-laying females (Abad et al. 2009; Moens et al. 2009 ). However, timing of RKN and gall development may differ depending on the host and nematode species. Papaya root sections showed localized swelling, with some GCs forming in the vascular cylinder from 9 DAI (Fig. 4 a,b). At this stage, initiating GCs resembled enlarged parenchymatic cells, reaching up to 4-times the normal size of cortical cells and characterized by multi-nucleation. Until now, it is not clear which vascular cells are GCs precursor from protophloem, pericycle, or protoxylem (Escobar et al. 2015 ). Nematode morphological changes, noted in the nematodes increasing girth, are characteristic of larval switch from migration to sedentary stage within established feeding sites (Williamson and Gleason, 2003 ). In Arabidopsis thaliana the first GCs induced by M. javanica develop by 3 DAI (Cabrera et al. 2015 ). Initiation of GC may occur as soon as 2 DAI in rice infested by M. graminicola (Nguyen et al. 2014; Petitot et al. 2017 ). M. incognita induce GCs from 4 DAI in rice and soybean (Fourie et al. 2013 ; Nguyen et al. 2014), and by 6 DAI in coffee ( Coffea arabica L.) (Albuquerque et al. 2010 ). Observation of initiating GCs around 9 DAI in papaya indicates that penetration and migration of M. javanica J2 probably took a longer time than other RKN species in other hosts. Juveniles secrete CAZymes produced by their subventral glands to soften root cell walls and facilitate their migration towards the vascular cylinder (Wieczorek, 2015 ). As observed for egg masses found inside the root cortex, production of CAZymes by M. javanica J2s may not allow rapid progression of larvae in papaya roots. By 16 DAI, the presence of "sausage-like" and saccate forms suggests the nematodes were transitioning through their second molt into the third juvenile stage (J3). Young M. javanica females were visible from 26 DAI corresponding to third-stage (J3) and fourth-stage (J4) juveniles transitioning into reproductive adults and most nematodes have developed into piriform shaped, egg-laying females at 35 DAI. The presence of egg masses with newly hatched juveniles and the persistence of feeding sites within the vascular cylinder confirm that nematodes continue to alter root structure as their population expands. Major advances was gained on the 3-D development of GCs within a gall by measurements of the volumes and shapes of the GCs induced by RKNs (Cabrera et al. 2015 ). We observed that GCs induced by M. javanica in papaya exhibited major enlargement between 9 and 16 DAI, along with thickened walls and increased vacuolization (Fig. 4 ). The enlargement apparently reached its maximum with GCs surface up to 94 000 µm 2 , more than 250-fold a normal vascular parenchymatic root cell (Fig. 6 ). GC surfaces measured in A. thaliana Col-0 could expand until 28 000 µm 2 with M. incognita (Vieira et al. 2013 ), or 40 000 µm 2 with M. javanica (Cabrera et al. 2015 ), probably because of different root sizes and architecture between papaya and Arabidopsis. In addition to mechanical restriction, the differential stimulation by the nematode could modify GC expansion during gall development as suggested by Cabrera et al. ( 2015 ) The proliferation of adjacent vascular and cortical cells was also evident in papaya galls. Notably, newly formed protophloem and xylem elements surrounded the GCs (Fig. 4 ). The hypertrophy of GCs and hyperplasia of surrounding tissue is a hallmark of root-knot nematode infections, as the plant vascular system is remodeled in galls to supply nutrients to the GCs that function as sinks for the parasite (Caillaud et al. 2008 ; Bartlem et al. 2013 ; Absmanner et al. 2013 ). In several host species infected by RKNs, spontaneous secondary root formation originating from the feeding site were reported (Nguyen et al. 2013), but we observed few lateral root formed on galls in papaya infested by M. javanica . Parallels between auxin-regulator players of lateral root formation and feeding site development were established (Cabrera et al. 2014 ). Histochemical staining techniques further elucidated the composition of the GCs and surrounding cells in papaya roots (Fig. 5 ). The NBB and PAS staining revealed that the cytoplasm of GCs contains high levels of proteins and polysaccharides, essential for sustaining the nematode metabolic needs. This finding is consistent with previous studies that indicated that galls and GCs have higher metabolic activity compared to healthy roots (Bird, 1961 ; Owens and Rubinstein, 1966 ; Gommers and Dropkin, 1977 ). Starch content increased three-fold in Medicago truncatula galls induced by M. incognita (Baldacci-Cresp et al. 2012 ). Transcriptome analyses of galls or dissected GCs reported the induction of the host primary metabolism (Jammes et al. 2005 ; Barcala et al. 2010 ; Kyndt et al. 2012 ; Ji et al. 2013 ; Petitot et al. 2017 ). Expression of sugar transport genes and the soluble sugar (fructose, glucose, sucrose) content were increased in tomato ( Solanum lycopersicum ) leaves and roots by RKN infection (Zhao et al. 2018 ). FASGA staining highlighted the presence of cellulose but did not detect lignin, suggesting that lignification is not a prominent feature in the thickened GC walls. This lack of lignification could facilitate the continuous nutrient transfer to the nematode, as lignified cell walls are typically more resistant to cellular modification. The staining results align with findings in other plant-parasitic nematodes, where GCs or syncytia are rich in proteins, polysaccharides, and other molecules required for nematode survival. In conclusion, the histological and histochemical analysis of papaya root infection by M. javanica demonstrates the extensive modifications induced by the nematode in root tissues, contributing to a broader understanding of the host-parasite interactions that underpin Meloidogyne infections in tropical crops. This study contributes valuable information for future research aiming at the development of resistant papaya cultivars and effective nematode management strategies. Declarations Author Contributions All authors contributed to the study conception and design. Material preparation and data collection were performed by KRLC, BT, MC, JS and DF. LC, KK and DF designed experiments, KRLC and DF analyzed data and wrote the manuscript. DF lead the work. The first draft of the manuscript was written by KRLC and DF and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Funding This research was funded by Institut de l’Environnement et de Recherches Agricoles (INERA, Burkina Faso) and the French Research Institute for Sustainable Development (IRD, France), and KRLC beneficiated from travel grants from the French Ministry of Foreign Affairs and IRD. Conflict of interest statement DF is an associate editor for this journal and the manuscript was independently handled by another member of the editorial board Data availability statement The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. 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CAB International, Wallingford, UK. pp 1-13 Nguyễn P, Bellafiore S, Petitot AS, Haidar R, Bak A, Abed A, Gantet P, Mezzalira I, de Almeida Engler J, Fernandez D (2014) Meloidogyne incognita - rice ( Oryza sativa ) interaction: a new model system to study plant–root-knot nematode interactions in monocotyledons. Rice 7(1) :23 https://doi.org/10.1186/s12284-014-0023-4 Owens RG, Rubinstein JH (1966) Metabolic changes induced by root-knot nematodes in host tissues. Contributions from Boyce Thompson Institute 23:199-214. Perry RN, Moens M, Starr FJ (2009) Root-Knot Nematodes. CAB International, Wallingford, UK. Petitot AS, Kyndt T, Haidar R, Dereeper A, Collin M, de Almeida Engler J, Gheysen G, Fernandez D (2017) Transcriptomic and histological responses of the African rice ( Oryza glaberrima ) to Meloidogyne graminicola provide new insights into root-knot nematode resistance in monocots. Annals of Botany 119: 885-899 DOI: 10.1093/aob/mcw256 Reversat G, Boyer J (1999) A mixture of sand and water-absorbent synthetic polymer as substrate for the xenic culturing of plant-parasitic nematodes in the laboratory. Nematology 1:209–212. Rutter WB, Franco J, Gleason C (2022) Rooting Out the Mechanisms of Root-Knot Nematode–Plant Interactions. Annual Review of Phytopathology 60:1–34 https://doi.org/10.1146/annurev-phyto-021621-120943 Triantaphyllou A, Hirschmann H (1960) Post-infection development of Meloidogyne incognita Chitwood (1949) Annales de l’Institut Phytopathologique Benaki, Kiphissia, Athens, Greece, 3:3–11 Tolivia D, Tolivia J (1987) Fasga - a new polychromatic method for simultaneous and differential staining of plant-tissues. J. Microscopy-Oxford 148: 113–117. doi: 10.1111/j.1365-2818.1987.tb02859.x Vieira P, Danchin EG, Neveu C, Crozat C, Jaubert S, Hussey RS, Engler G, Abad P, Almeida-Engler J, Castagnone-Sereno P, Rosso MN (2011) The plant apoplasm is an important recipient compartment for nematode secreted proteins. Journal of Experimental Botany 62:1241– 1253 https:// doi. org/ 10. 1093/ jxb/ erq352 Vieira P, Escudero C, Rodiuc N, Boruc J, Russinova E, Glab N, Mota M, De Veylder L, Abad P, Engler G, de Almeida Engler J (2013) Ectopic expression of Kip-related proteins restrains root-knot nematode-feeding site expansion. The New Phytologist, 199:505–519 https://doi.org/10.1111/nph.12255 Vilela RMIF, Kuster VC, Magalhães TA, Martini VC, Oliveira RM, de Oliveira DC (2023) Galls induced by a root-knot nematode in Petroselinum crispum (Mill.): impacts on host development, histology, and cell wall dynamics. Protoplasma 0123456789 https://doi.org/10.1007/s00709-023-01849-3 Wieczorek K (2015) Cell Wall Alterations in Nematode-Infected Roots. In: Escobar C, Fenoll C. (Eds) Advances in Botanical Research Plant Nematode Interactions: A View on Compatible Interrelationships. Academic Press, London, UK. pp 61-138 Williamson VM, Gleason CA (2003) Plant–nematode interactions. Current Opinion in Plant Biology 6:327–333 Zhao D, You Y, Fan H, Zhu X, Wang Y, Duan Y, Xuan Y, Chen L (2018) The Role of Sugar Transporter Genes during Early Infection by Root-Knot Nematodes. International Journal of Molecular Sciences19:302 doi: 10.3390/ijms19010302. Zijlstra C, Donkers-Venne DTHM, Fargette M (2000) Identification of Meloidogyne incognita , M. javanica and M. arenaria using sequence characterized amplified region (SCAR) based PCR assays. Nematology 2:847–853. Supplementary Files Table1Suppl.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Minor revisions 31 Jan, 2025 Reviewers agreed at journal 04 Dec, 2024 Reviewers invited by journal 04 Dec, 2024 Editor invited by journal 09 Nov, 2024 Editor assigned by journal 08 Nov, 2024 First submitted to journal 05 Nov, 2024 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-5377678","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":386152121,"identity":"de297479-9f99-4170-b4e3-76c565f5a2a2","order_by":0,"name":"Kouroubi R.L. COULIBALY","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kouroubi","middleName":"R.L.","lastName":"COULIBALY","suffix":""},{"id":386152122,"identity":"ffee8871-b5d9-4fdd-a26d-ee088abfcc9f","order_by":1,"name":"Bouma THIO","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Bouma","middleName":"","lastName":"THIO","suffix":""},{"id":386152123,"identity":"02af9ea4-73cf-4ea6-8dc9-2b5035c8fac4","order_by":2,"name":"Myriam COLLIN","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Myriam","middleName":"","lastName":"COLLIN","suffix":""},{"id":386152124,"identity":"3e76e471-9597-4799-ae6d-fe4bc08979ce","order_by":3,"name":"Kadidia KOITA","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kadidia","middleName":"","lastName":"KOITA","suffix":""},{"id":386152125,"identity":"d6a09f6b-dddf-4201-afab-d9f54c83708e","order_by":4,"name":"Jacob SANOU","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jacob","middleName":"","lastName":"SANOU","suffix":""},{"id":386152126,"identity":"0360448d-b45c-40aa-aa5a-551cd7fc60fa","order_by":5,"name":"Diana FERNANDEZ","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYDCCw2BSDsLhAbLYGBgMGCoIazGGazEGazmDT8sBNC2JDYS08B1nfvbgB4OBnPns5mcP3lTUpfdJH97AcHAPbi2Sh9nMDXsYDIxl7hwzN5xzhi23jS+tgOHAM9xaDA4zmEnwMPxJnCGRYCbN28aT28bDY8D84QA+LezfJP8wGNTPkEj/Js37TyKdDaiF4QBeLTxm0jwMBgkSEjlAWxoMEghqkTzMUyYtY2BgOEMip0xyzrEEwzYetoID+LTwnT++TfJNhYG8hET6Nok3NXXy8j3MGx/g0wJ1HhqfoIZRMApGwSgYBfgBANN5SNPT0qC9AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-1432-3129","institution":"Institut de recherche pour le developpement","correspondingAuthor":true,"prefix":"","firstName":"Diana","middleName":"","lastName":"FERNANDEZ","suffix":""}],"badges":[],"createdAt":"2024-11-02 10:18:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5377678/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5377678/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78935507,"identity":"04884c5c-4cf9-4906-951c-b814182a54ed","added_by":"auto","created_at":"2025-03-21 04:39:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":402709,"visible":true,"origin":"","legend":"\u003cp\u003ePapaya (\u003cem\u003eCarica papaya\u003c/em\u003e, variety Solo8) plantlets aged 4 weeks maintained in a growth chamber.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5377678/v1/7c931383a17283f76af10fab.png"},{"id":78935509,"identity":"abc45867-0532-4a7a-b39b-e699ac75c914","added_by":"auto","created_at":"2025-03-21 04:39:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1023574,"visible":true,"origin":"","legend":"\u003cp\u003eTypical developmental stages of \u003cem\u003eMeloidogyne javanica\u003c/em\u003e observed in papaya roots by staining nematodes with fuchsin at 9, 16, 26 and 35 days after inoculation (DAI). a) Second-stage juvenile (J2) at 9 DAI; b) Feeding J2 at 9 DAI ; c) Sausage-like feeding J2 at 16 DAI; d) J2 with saccate form and a spiked tail (black arrow) at 16 DAI; e) Young female at 26 DAI; F. Piriform mature female with an egg mass (em) at its posterior end laid in the root tissue.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5377678/v1/e8a0187984d4e4e5151f040a.png"},{"id":78935156,"identity":"ee1d5344-6bac-43df-b487-59074a22806c","added_by":"auto","created_at":"2025-03-21 04:31:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":81091,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMeloidogyne javanica\u003c/em\u003e developmental stages observed in papaya roots by staining nematodes with fuchsin at 9, 16, 26 and 35 days after inoculation (DAI). Two experiments were conducted with four plants at each time point inoculated with same initial densities of juveniles.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5377678/v1/ada3006ad37c96f35708c502.png"},{"id":78935608,"identity":"775cac35-948e-4ba3-b9fb-bea6f69354d2","added_by":"auto","created_at":"2025-03-21 04:47:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2060396,"visible":true,"origin":"","legend":"\u003cp\u003eHistological analysis of galls induced in papaya roots by \u003cem\u003eMeloidogyne javanica\u003c/em\u003e. Root (a, b) or gall (c, d, e, f) cross sections (10µm) were obtained at 9 days (a, b); 16 DAI (c, d); 26 DAI (e, f) after infection with the nematode and stained with toluidine blue (a, b) or Naphtol Blue Black- Periodic acid Schiff (c, d, e, f). One (a, c, d, e), two (b) or three (f) feeding sites are visible in the vascular cylinder with a nematode larva (N) in contact (a, c, d, e) with giant cells (*). Note the deep blue-stained nucleolus of nuclei in multi-nucleated giant cells. CP: cortical parenchyma; Ed: endodermis; Ep: epidermis; Pe: pericycle; VP: vascular parenchyma; Xy: xylem vessels; double arrow: proliferation of cell layers of parenchymatic origin.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5377678/v1/02fe2261de507ada40b1999e.png"},{"id":78935166,"identity":"76338a2c-4e91-4ddd-b2d2-f2d8ad5c6282","added_by":"auto","created_at":"2025-03-21 04:31:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1142002,"visible":true,"origin":"","legend":"\u003cp\u003eSuccessive cross sections (10µm) of a gall induced by \u003cem\u003eMeloidogyne javanica\u003c/em\u003e in papaya showing a feeding site\u003cem\u003e \u003c/em\u003emade of several giant cells (*)\u003cem\u003e \u003c/em\u003ewith a\u003cem\u003e \u003c/em\u003epiriform\u003cem\u003e \u003c/em\u003efemale nematode (N) and an egg mass (em) protruding outside the root at 35 days after inoculation. Sections were stained with either: a) Naphtol Blue Black- Periodic acid Schiff; b) FASGA; c) Periodic acid Schiff; d) DAPI; e)Toluidine Blue.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5377678/v1/ac8489851f74af7a2ff17e9d.png"},{"id":78935882,"identity":"ace85b45-860f-4140-8c4b-4b81e194240d","added_by":"auto","created_at":"2025-03-21 04:55:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":35945,"visible":true,"origin":"","legend":"\u003cp\u003eGiant cell surfaces (µm\u003csup\u003e2\u003c/sup\u003e) measured at different stages of \u003cem\u003eMeloidogyne javanica\u003c/em\u003e infection in papaya roots (9, 16, 26 and 35 days after inoculation (DAI)).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5377678/v1/9fef853ee0688e2cf481c239.png"},{"id":78935887,"identity":"ac539238-49a2-4076-8790-95e8c0f78ceb","added_by":"auto","created_at":"2025-03-21 05:03:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5429009,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5377678/v1/8b6da2dd-425f-4276-9788-d6c53aae9898.pdf"},{"id":78935158,"identity":"062544a4-4c83-4a6d-8c45-de9d59555c99","added_by":"auto","created_at":"2025-03-21 04:31:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13016,"visible":true,"origin":"","legend":"","description":"","filename":"Table1Suppl.docx","url":"https://assets-eu.researchsquare.com/files/rs-5377678/v1/ce45977b41645fac8bc49c5b.docx"}],"financialInterests":"","formattedTitle":"Life cycle of the Root-Knot Nematode Meloidogyne javanica in papaya and histological analyses of root infection and gall development.","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePapaya (\u003cem\u003eCarica papaya\u003c/em\u003e L.) is a tropical fruit crop grown in various tropical and subtropical regions around the world. Its rich nutritional value, unique flavor, and versatility have made it a popular choice among consumers worldwide. However, the successful cultivation of papaya is often hampered by various biotic and abiotic factors, with plant-parasitic nematodes emerging as one of the primary challenges. Nematodes are microscopic worms that inhabit the soil and attack the roots of plants. They inflict damage by feeding on the roots, causing root galling, stunting, and reduced nutrient uptake. The most common nematode species affecting papaya include the reniform nematode \u003cem\u003eRotylenchulus reniformis\u003c/em\u003e and root-knot nematodes (RKN) of the genus \u003cem\u003eMeloidogyne\u003c/em\u003e (Go\u0026euml;ldi 1892). A recent survey in Burkina Faso showed that RKNs (\u003cem\u003eMeloidogyne incognita\u003c/em\u003e and \u003cem\u003eMeloidogyne javanica\u003c/em\u003e) are commonly found in several regions of papaya cultivation (KRL Coulibaly and B Thio, pers. com.) These nematodes can drastically reduce fruit yield, leading to economic losses for farmers and impeding the growth of the papaya industry. Managing plant-parasitic nematodes in papaya cultivation necessitates an integrated approach combining cultural, biological, and chemical methods. Use of resistant/tolerant cultivars is a promising way to reduce damages caused by RKN (Kaloshian and Teixeira \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A breeding program is currently developed at INERA, Burkina to produce papaya varieties that can afford nematode infection. However, assessing nematode pathogenicity, and evaluating the efficacy of resistant/tolerant cultivars requires good knowledge about the life cycle of the nematodes into the roots. RKN are sedentary nematodes that settle in roots and feed on specialized cells during most stages of their life cycle that comprises five developmental stages separated by 4 molts (Tryantaphyllou and Hirschmann 1960; Eisenback \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Escobar et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The infective stage of \u003cem\u003eMeloidogyne\u003c/em\u003e is the second-stage juvenile (J2) that hatch from eggs in soil and is specifically attracted by root exudates towards host plant roots. J2s can directly penetrate the root apex and migrate through the cortex, moving between cells, to reach the vascular tissues. The nematode use a combination of mechanical piercing through its stylet and cell wall softening by producing specific carbohydrates degrading enzymes (CAZymes) to enter and migrate into roots (Wieczorek \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Upon reaching the vascular cylinder, J2s settle and start feeding on certain parenchyma cells. Salivary secretions produced by the nematode induce the transformation of the parenchyma cells into hypertrophied and multinucleated \"giant cells\" (GC) encaged within a network of \u003cem\u003ede novo\u003c/em\u003e formed xylem and phloem cells which serve as active feeding sites for the nematode (Bartlem et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In addition, GC formation is accompanied by proliferation of adjacent cells resulting in visible root deformation known as gall, disrupting the normal root architecture and growth (Kyndt et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Sedentary nematodes within galls feed on giant cells (GC) to progress through their larval development from the third-stage (J3) and fourth-stage (J4) to adulthood. Sexual differentiation into males or females may occur based on environmental conditions. Under unfavorable conditions, males may develop; however, in most \u003cem\u003eMeloidogyne\u003c/em\u003e species, females that reproduce parthenogenetically are typically produced. They then lay eggs in a gelatinous matrix outside the root, with exception of \u003cem\u003eMeloidogyne graminicola\u003c/em\u003e that lay eggs inside the root cortex of rice (\u003cem\u003eOryza sativa\u003c/em\u003e L.) (Fernandez et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The larvae that hatch from these eggs can reinfect the same root or migrate into the soil to infect other plants.\u003c/p\u003e \u003cp\u003eThe ability of \u003cem\u003eMeloidogyne\u003c/em\u003e to manipulate plant cell metabolism to create their feeding site, without activating the innate immune system of their host is a key factor in their parasitic success (Rutter et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In resistant plants, the RKN life cycle may be stopped at pre-infection stages preventing juveniles attraction and penetration or in subsequent post-infection stages impeding GC formation or development until female production (Goverse and Smant \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Goode and Mitchum \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe histology of RKN \u0026ndash; host interaction has been described in several plant species highlighting the specific features of GC and gall development in susceptible and resistant plants (for review Escobar et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Host immune reactions include different mechanisms underlying basal and specific nematode resistance, including callose deposition and typical hypersensitive responses (HR) blocking the nematode in an early stage (Albuquerque et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) or the feeding structure collapses because of nuclear and cytoplasmic degeneration (Petitot et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRKN development in papaya roots was never examined. In the field, the variety Solo is susceptible to RKN. We present, for the first time, an analysis of RKN development in papaya roots and the histological alterations induced in host tissues. This investigation utilized a combination of light microscopy techniques, including the observation of fuchsin-stained nematodes and a detailed histological analysis of root galls and GC induced by the nematode.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003eCulture and inoculation of nematodes\u003c/p\u003e \u003cp\u003eA population of \u003cem\u003eMeloidogybe javanica\u003c/em\u003e was isolated from an egg mass taken from papaya roots of the FBPA1 variety in the INERA Farako B\u0026acirc; research station, (4\u0026deg;20'W; 11\u0026deg;06'N) near the town of Bobo-Dioulasso, Burkina Faso. The identification of the species \u003cem\u003eM. javanica\u003c/em\u003e was confirmed following molecular characterization using the specific SCAR marker developed by Zijlstra et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The multiplication of the nematode was carried out on tomato plants (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e L. cv. Naine) in a culture chamber (26\u0026deg;C/24\u0026deg;C day/night temperature, under a 14-h day/10-h night light regime with a Photosynthetic Photon Flux Density (PPFD) of 210 \u0026micro;mol/s.m2) with 78% relative humidity (RH) at Institut de Recherche pour le D\u0026eacute;veloppement (IRD), Montpellier, France. Three months after inoculation, the nematode eggs were recovered by immersing the roots in 0.5% sodium hypochlorite for 3 minutes before mixing them in a blender for 5 times during 1s. Eggs were collected onto a 25 \u0026micro;m mesh and were hatched at 28\u0026deg;C. Second stage juveniles (J2) collected after a maximum of 72 h were counted and used as inoculum.\u003c/p\u003e \u003cp\u003eCultivation of papaya trees and inoculation of \u003cem\u003eM. javanica\u003c/em\u003e\u003c/p\u003e \u003cp\u003ePapaya seeds of the cultivated Solo variety (n\u0026deg;8 Technisem lot n\u0026deg; 31072-0820-98) were germinated on sand wet with Hoagland's solution (diluted 4 times) (KNO3 5 mM; KH2PO4 1 mM; Ca(NO3)2 5 mM; MgSO4 2 mM; 25 mg iron (Fe-EDDHA); trace elements) and maintained in a culture chamber (same conditions as for nematodes). After germination (3\u0026ndash;4 weeks long), the papaya seedlings were transferred to a 26 mm-diameter PVC tube (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) filled with SAP (sand and water-absorbent synthetic polymer) (Reversat et al. 1999) and watered 3 times per week with Hoagland's solution (diluted 4 times). Papaya plantlets were inoculated after 3\u0026ndash;4 weeks of growth by carefully opening the SAP cylinder contained into the PVC tube and bringing an aqueous suspension containing the J2 of \u003cem\u003eM. javanica\u003c/em\u003e all along the exposed root system using a micropipette. At this stage, papaya root systems were ca. 15-20-cm long and several lateral roots arised from the main root. Two inoculation assays were conducted (on June 15 and June 20, 2022). In the first assay, papaya seedlings were inoculated with 400 \u003cem\u003eM. javanica\u003c/em\u003e J2, followed by the second assay in which seedlings were each inoculated with 2000 J2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMonitoring the infectious process by fuschin staining\u003c/p\u003e \u003cp\u003ePapaya roots infested with \u003cem\u003eM. javanica\u003c/em\u003e were collected at 9, 16, 26 and 35 days after inoculation (DAI), stained by immersing during 1 min in boiling acid fuschin, rinsed with osmosis water and placed in acidified glycerol according to the method of Byrd et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). Roots were first observed under a binocular loop, and the infested parts were dissected and mounted in glycerol between slide and coverslip. Nematode developmental stages (J2, feeding or not, young females, mature females and eggs) were assessed under the Leica DM1000 microscope (40x and 100x magnification) following determination of Tryantaphyllou and Hirschmann (1960). However, we could not determine the sexual stage of feeding J2 nor the J3 and J4 stages with precision. Typical developmental stages were recorded and photographed to take nematode measures (length and width). The Zeiss Axio Imager Z2 microscope equipped with an Axiocam 506 camera was used to take the photos.\u003c/p\u003e \u003cp\u003eHistological analysis of the infection of papaya roots\u003c/p\u003e \u003cp\u003eSome root tip fragments of 0.5 to 1 cm or ca. 2\u0026ndash;4 visible galls on the root system were randomly taken from plants used for fuchsin staining at 9, 16, 26 and 35 DAI. Samples from each plant were processed separately during all stages, embedded in separate blocks and histological data were recorded from sections obtained from each block at each time point. Root and gall samples were immediately immersed in a fixative (2% paraformaldehyde, 1% glutaraldehyde and 1% caffeine), infiltrated under vacuum for 15 minutes and incubated at 4\u0026deg;C for 3 days. Then, the samples were dehydrated in 30% and 50% ethanol for 1 h each and then kept in 70% ethanol. The dehydration of the samples continued in ethanol at increasing concentration (80%, 90%, 95% and 100%) for 1 hour at room temperature before storage in 100% ethanol overnight at 4\u0026deg;C. The fixed root and gall samples were embedded in resin (epoxy Technovit 7100, Kulzer Friedrichsdorf, Germany) and blocks were sectioned using the Epredia\u0026trade; HM 355S microtome (Fischer Thermo Scientific, France). Sections (10 \u0026micro;m width) were mounted in slides (3 sections per slide) and examined without staining to detect large cells typical of feeding sites. Sections with feeding sites were initially stained with 0.05% toluidine blue in 0.1 M sodium phosphate buffer (PBS), pH 5.5 (3 min at room temperature) for cytological observations. To detect soluble proteins in cells, sections were stained with Naphthol Blue Black (NBB). Polysaccharides were detected with PAS, for Periodic Acid 2% for 5 minutes followed by Schiff 1% in PBS for 15 minutes in the dark. Lignins and cellulose were detected with Fasga (diluted 1/7 in PBS for 3 h), and nucleic acids with Dapi (1/100 in PBS for 5 minutes). After staining, the sections were dried and mounted between slide and coverslip for microscopic observations. Images were taken using the Zeiss Axio Imager Z2 microscope and ZEN 3.6 blue edition software.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eNematode cycle in papaya roots\u003c/h2\u003e \u003cp\u003eThe developmental cycle of \u003cem\u003eM. javanica\u003c/em\u003e was assessed by staining the nematodes in roots with acid fuchsin at 9, 16, 26, and 35 DAI. This time course was arbitrarily determined based on preliminary trials with a small number of plants. Two assays were conducted with four plants inoculated with same initial densities (ID) of juveniles of \u003cem\u003eM. javanica\u003c/em\u003e analysed at each time point, and nematodes were observed and counted in roots. Depending on the number of nematodes inoculated on each root system, an average (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD; \u003cem\u003en\u0026thinsp;=\u0026thinsp;12\u003c/em\u003e) of 65\u0026thinsp;\u0026plusmn;\u0026thinsp;28 (ID\u0026thinsp;=\u0026thinsp;400 J2) to 177\u0026thinsp;\u0026plusmn;\u0026thinsp;104 (ID\u0026thinsp;=\u0026thinsp;2000 J2) juveniles successfully penetrated and established in papaya roots. On average, 32 (ID\u0026thinsp;=\u0026thinsp;400 J2) to 62 (ID\u0026thinsp;=\u0026thinsp;2000 J2) galls formed on each root system. Typical nematode developmental stages observed are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt day 9, papaya roots exhibited slight swellings, with barely visible galls, especially at the root tips. Nematodes were distributed along the root system, from tip to 10 cm up. Inside roots, larvae were mostly found near or inside the stele. \u003cem\u003eM. javanica\u003c/em\u003e larvae remained vermiform typical of the J2 infective stage either filiform (15\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026micro;m in width; 378\u0026thinsp;\u0026plusmn;\u0026thinsp;28 \u0026micro;m in length, \u003cem\u003en\u0026thinsp;=\u0026thinsp;19\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), or showing a slight increase in width (25\u0026thinsp;\u0026plusmn;\u0026thinsp;6 \u0026micro;m, \u003cem\u003en\u0026thinsp;=\u0026thinsp;19\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), suggesting that feeding on plant has started.\u003c/p\u003e \u003cp\u003eAt day 16, visible galls had formed and were scattered throughout the root system. Nematodes located within the galls were predominantly found in the cortex, oriented with their heads toward the vascular cylinder, and had increased in thickness without a corresponding increase in length, indicating active feeding on the plant. Some feeding J2 remained vermiform, similar in size to those observed at 9 DAI (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), while others had developed a swollen, \"sausage-like\" shape (56\u0026thinsp;\u0026plusmn;\u0026thinsp;8 \u0026micro;m in width, \u003cem\u003en\u0026thinsp;=\u0026thinsp;10\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Additionally, several nematodes exhibited a more saccate form with a spiked tail, suggesting they were shortly before the second molt transitioning into the third-stage juvenile (J3) (82\u0026thinsp;\u0026plusmn;\u0026thinsp;17 \u0026micro;m in width; 381\u0026thinsp;\u0026plusmn;\u0026thinsp;41 \u0026micro;m in length, \u003cem\u003en\u0026thinsp;=\u0026thinsp;12\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eAt day 26, larger galls developed throughout the root system. Three \u003cem\u003eM. javanica\u003c/em\u003e developmental stages were observed, with nematodes either close to stages J3 as at day 16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), or young females with rounded posterior end (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee) or pear-shaped mature females (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). No male was observed. Female body had considerably increased in size. Young females measured 191\u0026thinsp;\u0026plusmn;\u0026thinsp;59 \u0026micro;m large (posterior end) and 515\u0026thinsp;\u0026plusmn;\u0026thinsp;90 \u0026micro;m long (\u003cem\u003en\u0026thinsp;=\u0026thinsp;14\u003c/em\u003e), and mature females reached 312\u0026thinsp;\u0026plusmn;\u0026thinsp;53 large (posterior end) and 579\u0026thinsp;\u0026plusmn;\u0026thinsp;75 long (\u003cem\u003en\u0026thinsp;=\u0026thinsp;17\u003c/em\u003e). Feeding sites with pink-stained GCs were observed near the female heads embedded in the root vascular cylinder.\u003c/p\u003e \u003cp\u003eAt day 35, most \u003cem\u003eM. javanica\u003c/em\u003e observed in galls were mature females with a pyriform shape and an egg mass attached to their posterior end (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Some egg masses protruded outside the papaya root but the majority of them remained enclosed in the cortical root tissue. Female size was up to 446\u0026thinsp;\u0026plusmn;\u0026thinsp;76 \u0026micro;m large (posterior end) and 723\u0026thinsp;\u0026plusmn;\u0026thinsp;104 long (\u003cem\u003en\u0026thinsp;=\u0026thinsp;43\u003c/em\u003e). Newly hatched J2s of the next generation were also observed in vicinity of egg masses.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e summarizes the two experiments showing the percentage of nematodes at different developmental stages (infective J2, young females, mature females, and newly hatched J2) during the infection of papaya roots by \u003cem\u003eM. javanica\u003c/em\u003e. In both experiments (1 and 2), J2 stages accounted for 100% of the population at 9 days DAI and over 99% at 16 DAI indicating that the initial penetration and establishment phases were synchronous across experiments. The majority of immature young females were observed starting at 26 DAI, making up more than 80% of the total population. By 35 DAI, only mature females with eggs were observed, alongside approximately 10\u0026ndash;15% of newly hatched J2. These data show that the \u003cem\u003eM. javanica\u003c/em\u003e cycle in papaya, from J2 penetration to the next generation J2 release, has a duration of approximately 35 DAI at 28\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHistological analysis of the infection of papaya roots with\u003c/b\u003e \u003cb\u003eM. javanica\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRoot tips and galls were collected at different times after inoculation to highlight the histological structure of the feeding sites induced by \u003cem\u003eM. javanica\u003c/em\u003e in papaya. Examination of longitudinal root sections and transverse gall sections stained with toluidine blue or NBB-PAS allowed observation of the morphology of GCs and their subcellular components, as well as of the surrounding cells at different stages post-infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Several sections of at least 20 feeding sites were observed at each time point.\u003c/p\u003e \u003cp\u003eAt day 9, histological blocks contained mostly swollen root tips and few galls. Longitudinal sections of roots showed well structured cell layers, including epidermis, cortical parenchyma, pericycle, endodermis and vascular parenchyma (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b). Several hypertrophied vascular parenchymatic cells characteristic of GCs are found associated to J2 inside the root vascular cylinder close to the meristeme or higher on the root (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b). Approximately 4 to 7 GCs per feeding site are visible on each root section plane. GC are multinucleated, with 4\u0026ndash;10 nuclei with large dense nucleolus (deep-blue stained) on each cell section indicating that intense DNA replication was taking place. The cytoplasm of giant cells appears dense and contains 1\u0026ndash;2 large vacuoles. A discrete proliferation of vascular parenchymatic cells adjacent to the CGs is observed. The nematodes visible on the sections had increased in size compared to the inoculated J2. Importantly, all the feeding sites observed were located inside the vascular cylinder, well delimited by and no feeding sites was found in the root cortex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt day 16, several feeding sites with swollen feeding nematodes are observed in all sectioned galls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d). The CGs are housed in the vascular cylinder that conserved its integrity despite swelling. GCs had largely increased in size compared to day 9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). They present thick walls and their dense, granular-looking cytoplasm contains several small vacuoles. Several nuclei with large dense nucleolus are well visible. Around 4 to 8 CGs are observed per feeding site and look embedded with newly formed xylem vessels and phloem elements. Xylem elements can be identified by the thick cells walls, differential toluidine blue or NBB staining, and banding patterns of lignified cell wall thickenings (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, e, f). In contrast, the phloem appears as sieve elements that are frequently nucleated.\u003c/p\u003e \u003cp\u003eIncreased proliferation of adjacent cells into several (4\u0026ndash;5) layers was observed from both sides of GCs in longitudinal sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The newly formed cells apparently originated from the pericycle in vascular cylinder as well as from the root cortex. The new vascular parenchymatic cells, probably phloem elements, lost their typical rectangular shape and presented irregular shapes encircling the giant-feeding cells. All nematodes retained their elongated form, likely representing J2s. Nematode head was embedded among CGs while their body lied inside the vascular parenchyma (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eAt day 26, several well-developed feeding sites were observed near the nematodes within the vascular cylinder, all visible in the same cross-sectional plane (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, f). GCs with thickened walls, asymmetrical shapes, numerous protrusions, and dense cytoplasm containing few vacuoles were noted. On NBB-PAS-stained sections, the blue staining of the giant cell cytoplasm, along with the proliferation of pink-stained parenchymal cells, became more pronounced at this stage of development. Feeding sites appear well delimited in the stele that gained 4\u0026ndash;5 additional surrounding cell layers. Some nematodes displayed a rounded shape characteristic of young females (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee), suggesting that they underwent molts (J2-J3-J4-young females). Sometimes, the induction of lateral roots originating from pericycle cells was also visible situated at the feeding site.\u003c/p\u003e \u003cp\u003eAt day 35, numerous feeding sites were observed within the vascular cylinder, accompanied by increased proliferation of adjacent cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The GCs were generally not visibly different from those formed 26 DAI. The majority of nematodes were mature egg-laying females with egg masses in a gelatinous matrix deposited either inside the cortex or outside the cortex. Females had their posterior body part, now of piriform shape, located in the cortex, and a long anterior \u0026lsquo;neck\u0026rsquo; and head embedded in a group of GCs located in the vascular cylinder.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe surface area of GCs was measured on the two to three largest GCs per feeding site, with 54 giant cell sections recorded at each time point (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). On day 9, the maximum GC surface area measured in a section was 22,688 \u0026micro;m\u0026sup2;. A substantial increase in GC surface area was observed by 16 DAI, with the highest recorded value reaching 94,781 \u0026micro;m\u0026sup2;. No further increase in GC surface area was noted at subsequent time points. In comparison, the surface areas of vascular parenchymatic and cortical cells, measured in the same sections, showed no significant variation over time, with averages of 348\u0026thinsp;\u0026plusmn;\u0026thinsp;59 \u0026micro;m\u0026sup2; and 5,227\u0026thinsp;\u0026plusmn;\u0026thinsp;1,243 \u0026micro;m\u0026sup2;, respectively (Table\u0026nbsp;1, supplemental data).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGCs function as nutrient source tissues, supplying nematodes with soluble sugars and other essential molecules transported from the aerial parts of the plant. Sections were stained using a combination of NBB and PAS dyes. NBB stains proteins blue, while PAS highlights polysaccharides (glycogen, starch or cellulose) in pink. In all NBB-PAS-stained sections presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, both the nematode and GC cytoplasm appeared uniformly blue, with the thickened walls of GCs and other root cell walls stained a deep pink. Nuclei in all cells presented a dark blue nucleolus, indicating a high protein content probably reflecting the ribosome biosynthesis activity. Additionally, DAPI dye characteristic of nucleic acids confirmed nuclei identification in GC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). When PAS alone was applied, polysaccharides were revealed in cell walls (cellulose) and GCs cytoplasm was stained as uniformly pale pink (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), indicative of weak presence of glycogen or starch, but no starch grain were visible.\u003c/p\u003e \u003cp\u003eTo further investigate the gall tissue structure and cell wall components, some sections were stained using FASGA, a mixture of fuchsin, alcian blue, safranin, and glycerin, which stains cellulose (and pectin) blue-green and lignin red (Tolivia and Tolivia, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). FASGA stained all root cell walls blue-green, including the thickened GC walls, and did not reveal lignin deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Red staining was only observed in certain deposits within xylem vessels (data not shown). The nematode itself did not stain with either FASGA dye.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we assessed the key developmental stages of \u003cem\u003eM. javanica\u003c/em\u003e in papaya (variety Solo) roots during 35 days. In addition, the histological examination of papaya roots during infection provided crucial insights into the dynamic interactions between the nematode and the host plant. The analysis highlights how the development of feeding sites, particularly GCs, progresses over time and the impact of this process on root tissue architecture. These findings are consistent with previous studies on RKN infecting other host plants (Perry et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) yet exhibiting some differences and contribute to a broader understanding of plant-nematode interactions.\u003c/p\u003e \u003cp\u003e \u003cem\u003eM. javanica\u003c/em\u003e cycle in papaya lasted 5 weeks in our culture chamber conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). By 35 DAI, the nematodes were all mature females, and egg masses were observed in galls. The large size of piriform females (up to 723 \u0026micro;m in length) and the presence of newly hatched J2s in the vicinity of egg masses confirmed the completion of the reproductive cycle. This observation mirrors findings in other studies that note the \u003cem\u003eMeloidogyne\u003c/em\u003e life cycle typically culminates in the production of egg masses within 4\u0026ndash;5 weeks under optimal conditions (Bird, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1959\u003c/span\u003e; Dropkin, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1963\u003c/span\u003e). \u003cem\u003eM. javanica\u003c/em\u003e has a large host range and is commonly found in tropical areas (Evans and Perry, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In rice (\u003cem\u003eO. sativa\u003c/em\u003e), the \u003cem\u003eM. javanica\u003c/em\u003e cycle has a similar duration with all females laying eggs at 28 DAI in the same experimental conditions (Grossi de S\u0026aacute; et al. 2019). Interestingly, several \u003cem\u003eM. javanica\u003c/em\u003e egg masses were found inside the root cortex of papaya (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This feature is common in \u003cem\u003eM. graminicola\u003c/em\u003e, a RKN species that attacks several graminaceous hosts including rice, but was never reported for \u003cem\u003eM. javanica\u003c/em\u003e. \u003cem\u003eM. javanica\u003c/em\u003e can attack rice grown in rainfed systems but produce eggs outside the root (Grossi de S\u0026aacute; et al. 2019). Egg-laying in the cortex has been thought to be an adaptation of \u003cem\u003eM. graminicola\u003c/em\u003e to flooding conditions in irrigated rice fields. However, this is not the case for papaya cultivation and it is questioning whether it could be an adaptation to this particular plant species. Nematode females produce \u003cem\u003evia\u003c/em\u003e vulval secretions some plant cell wall modifying proteins for the breakdown of root cells in order to adjust space for their body enlargement and likely to expulse their eggs at root surface (Vieira et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). It is therefore possible that in some strong papaya roots \u003cem\u003eM. javanica\u003c/em\u003e females will not be able to produce enough degrading secretions to expulse their eggs. The presence of hidden \u003cem\u003eM. javanica\u003c/em\u003e egg masses inside roots is an important data that breeders should take into account when screening papaya varieties for resistance to RKNs. Egg mass production on roots is frequently used as a parameter for plant resistance to nematodes that may be underestimated in the case of papaya \u0026ndash; \u003cem\u003eM. javanica\u003c/em\u003e interactions.\u003c/p\u003e \u003cp\u003eThe infection process of \u003cem\u003eM. javanica\u003c/em\u003e in papaya roots followed a well-documented developmental timeline of RKNs, characterized by the formation of galls, the establishment of GCs in the stele, and the maturation of nematodes into egg-laying females (Abad et al. 2009; Moens et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). However, timing of RKN and gall development may differ depending on the host and nematode species. Papaya root sections showed localized swelling, with some GCs forming in the vascular cylinder from 9 DAI (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b). At this stage, initiating GCs resembled enlarged parenchymatic cells, reaching up to 4-times the normal size of cortical cells and characterized by multi-nucleation. Until now, it is not clear which vascular cells are GCs precursor from protophloem, pericycle, or protoxylem (Escobar et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Nematode morphological changes, noted in the nematodes increasing girth, are characteristic of larval switch from migration to sedentary stage within established feeding sites (Williamson and Gleason, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eArabidopsis thaliana\u003c/em\u003e the first GCs induced by \u003cem\u003eM. javanica\u003c/em\u003e develop by 3 DAI (Cabrera et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Initiation of GC may occur as soon as 2 DAI in rice infested by \u003cem\u003eM. graminicola\u003c/em\u003e (Nguyen et al. 2014; Petitot et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eM. incognita\u003c/em\u003e induce GCs from 4 DAI in rice and soybean (Fourie et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Nguyen et al. 2014), and by 6 DAI in coffee (\u003cem\u003eCoffea arabica\u003c/em\u003e L.) (Albuquerque et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Observation of initiating GCs around 9 DAI in papaya indicates that penetration and migration of \u003cem\u003eM. javanica\u003c/em\u003e J2 probably took a longer time than other RKN species in other hosts. Juveniles secrete CAZymes produced by their subventral glands to soften root cell walls and facilitate their migration towards the vascular cylinder (Wieczorek, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). As observed for egg masses found inside the root cortex, production of CAZymes by \u003cem\u003eM. javanica\u003c/em\u003e J2s may not allow rapid progression of larvae in papaya roots.\u003c/p\u003e \u003cp\u003eBy 16 DAI, the presence of \"sausage-like\" and saccate forms suggests the nematodes were transitioning through their second molt into the third juvenile stage (J3). Young \u003cem\u003eM. javanica\u003c/em\u003e females were visible from 26 DAI corresponding to third-stage (J3) and fourth-stage (J4) juveniles transitioning into reproductive adults and most nematodes have developed into piriform shaped, egg-laying females at 35 DAI. The presence of egg masses with newly hatched juveniles and the persistence of feeding sites within the vascular cylinder confirm that nematodes continue to alter root structure as their population expands.\u003c/p\u003e \u003cp\u003eMajor advances was gained on the 3-D development of GCs within a gall by measurements of the volumes and shapes of the GCs induced by RKNs (Cabrera et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). We observed that GCs induced by \u003cem\u003eM. javanica\u003c/em\u003e in papaya exhibited major enlargement between 9 and 16 DAI, along with thickened walls and increased vacuolization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The enlargement apparently reached its maximum with GCs surface up to 94 000 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e, more than 250-fold a normal vascular parenchymatic root cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). GC surfaces measured in \u003cem\u003eA. thaliana\u003c/em\u003e Col-0 could expand until 28 000 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e with \u003cem\u003eM. incognita\u003c/em\u003e (Vieira et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), or 40 000 \u0026micro;m\u003csup\u003e2\u003c/sup\u003ewith \u003cem\u003eM. javanica\u003c/em\u003e (Cabrera et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), probably because of different root sizes and architecture between papaya and Arabidopsis. In addition to mechanical restriction, the differential stimulation by the nematode could modify GC expansion during gall development as suggested by Cabrera et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe proliferation of adjacent vascular and cortical cells was also evident in papaya galls. Notably, newly formed protophloem and xylem elements surrounded the GCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The hypertrophy of GCs and hyperplasia of surrounding tissue is a hallmark of root-knot nematode infections, as the plant vascular system is remodeled in galls to supply nutrients to the GCs that function as sinks for the parasite (Caillaud et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Bartlem et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Absmanner et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In several host species infected by RKNs, spontaneous secondary root formation originating from the feeding site were reported (Nguyen et al. 2013), but we observed few lateral root formed on galls in papaya infested by \u003cem\u003eM. javanica\u003c/em\u003e. Parallels between auxin-regulator players of lateral root formation and feeding site development were established (Cabrera et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHistochemical staining techniques further elucidated the composition of the GCs and surrounding cells in papaya roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The NBB and PAS staining revealed that the cytoplasm of GCs contains high levels of proteins and polysaccharides, essential for sustaining the nematode metabolic needs. This finding is consistent with previous studies that indicated that galls and GCs have higher metabolic activity compared to healthy roots (Bird, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1961\u003c/span\u003e; Owens and Rubinstein, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1966\u003c/span\u003e; Gommers and Dropkin, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). Starch content increased three-fold in \u003cem\u003eMedicago truncatula\u003c/em\u003e galls induced by \u003cem\u003eM. incognita\u003c/em\u003e (Baldacci-Cresp et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Transcriptome analyses of galls or dissected GCs reported the induction of the host primary metabolism (Jammes et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Barcala et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Kyndt et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Ji et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Petitot et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Expression of sugar transport genes and the soluble sugar (fructose, glucose, sucrose) content were increased in tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) leaves and roots by RKN infection (Zhao et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFASGA staining highlighted the presence of cellulose but did not detect lignin, suggesting that lignification is not a prominent feature in the thickened GC walls. This lack of lignification could facilitate the continuous nutrient transfer to the nematode, as lignified cell walls are typically more resistant to cellular modification. The staining results align with findings in other plant-parasitic nematodes, where GCs or syncytia are rich in proteins, polysaccharides, and other molecules required for nematode survival.\u003c/p\u003e \u003cp\u003eIn conclusion, the histological and histochemical analysis of papaya root infection by \u003cem\u003eM. javanica\u003c/em\u003e demonstrates the extensive modifications induced by the nematode in root tissues, contributing to a broader understanding of the host-parasite interactions that underpin \u003cem\u003eMeloidogyne\u003c/em\u003e infections in tropical crops. This study contributes valuable information for future research aiming at the development of resistant papaya cultivars and effective nematode management strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation and data collection were performed by KRLC, BT, MC, JS and DF. LC, KK and DF designed experiments, KRLC and DF analyzed data and wrote the manuscript. DF lead the work. The first draft of the manuscript was written by KRLC and DF and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by Institut de l’Environnement et de Recherches Agricoles (INERA, Burkina Faso) and the French Research Institute for Sustainable Development (IRD, France), and KRLC beneficiated from travel grants from the French Ministry of Foreign Affairs and IRD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDF is an associate editor for this journal and the manuscript was independently handled by another member of the editorial board\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbsmanner B, Stadler R, Hammes UZ (2013) Phloem development in nematode- induced feeding sites: the implications of auxin and cytokinin. Frontiers in Plant Science 4:241 \u003c/li\u003e\n\u003cli\u003eAlbuquerque EVS, Carneiro RMDG, Costa PM, GomesACMM, Santos M, Pereira AA, Nicole M, Fernandez D, Grossi-de-Sa MF (2010) Resistance to \u003cem\u003eMeloidogyne incognita\u003c/em\u003e expresses a hypersensitive-like response in \u003cem\u003eCoffea arabica\u003c/em\u003e. European Journal of Plant Pathology 127:365\u0026ndash;373 https://doi.org/10.1007/s10658-010-9603-3\u003c/li\u003e\n\u003cli\u003eBaldacci-Cresp F, Chang C, Maucourt M, Deborde C, Hopkins J, Lecomte P, et al (2012) (Homo)glutathione deficiency impairs root-knot nematode development in \u003cem\u003eMedicago truncatula\u003c/em\u003e. PloS Pathogens, 8(1), https://doi.org/10.1371/journal.ppat.1002471\u003c/li\u003e\n\u003cli\u003eBarcala M, Garcia A, Cabrera J, Casson S, Lindsey K, Favery B, Garcia-Casado G, Solano R, Fenoll C, Escobar C (2010) Early transcriptomic events in microdissected \u003cem\u003eArabidopsis\u003c/em\u003e nematode- induced giant cells. Plant J 61:698\u0026ndash;712, DOI: 10.1111/j.1365-313X.2009.04098.x\u003c/li\u003e\n\u003cli\u003eBartlem DG, Jones MGK, Hammes UZ (2013) Vascularization and nutrient delivery at root-knot nematode feeding sites in host roots. Journal of Experimental Botany 65:1789\u0026ndash;1798 https://doi.org/10.1093/jxb/ert415\u003c/li\u003e\n\u003cli\u003eBird AF (1959) Development of the root-knot nematodes \u003cem\u003eMeloidogyne javanica\u003c/em\u003e Treub and \u003cem\u003eMeloidogyne hapla\u003c/em\u003e Chitwood in the tomato. 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Planta, 238:807\u0026ndash;818 https://doi.org/10.1007/s00425-013-1923-z\u003c/li\u003e\n\u003cli\u003eMoens M, Perry RN, Starr FJ (2009) \u003cem\u003eMeloidogyne\u003c/em\u003e species \u0026ndash; a diverse group of novel and important plant parasites. In: Perry RN, Moens M, Starr FJ (Eds) Root-Knot Nematodes. CAB International, Wallingford, UK. pp 1-13\u003c/li\u003e\n\u003cli\u003eNguyễn P, Bellafiore S, Petitot AS, Haidar R, Bak A, Abed A, Gantet P, Mezzalira I, de Almeida Engler J, Fernandez D (2014) \u003cem\u003eMeloidogyne incognita\u003c/em\u003e - rice (\u003cem\u003eOryza sativa\u003c/em\u003e) interaction: a new model system to study plant\u0026ndash;root-knot nematode interactions in monocotyledons. Rice 7(1) :23 https://doi.org/10.1186/s12284-014-0023-4\u003c/li\u003e\n\u003cli\u003eOwens RG, Rubinstein JH (1966) Metabolic changes induced by root-knot nematodes in host tissues. Contributions from Boyce Thompson Institute 23:199-214. \u003c/li\u003e\n\u003cli\u003ePerry RN, Moens M, Starr FJ (2009) Root-Knot Nematodes. CAB International, Wallingford, UK. \u003c/li\u003e\n\u003cli\u003ePetitot AS, Kyndt T,\u003csup\u003e \u003c/sup\u003eHaidar R, Dereeper A, Collin M, de Almeida Engler J, Gheysen G, Fernandez D (2017) Transcriptomic and histological responses of the African rice (\u003cem\u003eOryza glaberrima\u003c/em\u003e) to \u003cem\u003eMeloidogyne\u003c/em\u003e \u003cem\u003egraminicola\u003c/em\u003e provide new insights into root-knot nematode resistance in monocots. Annals of Botany 119: 885-899 DOI: 10.1093/aob/mcw256\u003c/li\u003e\n\u003cli\u003eReversat G, Boyer J (1999) A mixture of sand and water-absorbent synthetic polymer as substrate for the xenic culturing of plant-parasitic nematodes in the laboratory. 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Nematology 2:847\u0026ndash;853.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"tropical-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tppa","sideBox":"Learn more about [Tropical Plant Pathology](https://www.springer.com/journal/40858)","snPcode":"40858","submissionUrl":"https://www.editorialmanager.com/tppa","title":"Tropical Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Carica papaya, gall, giant cell, histology, microscopy, nematology","lastPublishedDoi":"10.21203/rs.3.rs-5377678/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5377678/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePapaya (\u003cem\u003eCarica papaya\u003c/em\u003e L.) is susceptible to attacks by root-knot nematodes (RKN), which lead to significant production losses. Understanding the life cycle of RKN in papaya is essential for developing effective control strategies and screening for natural resistance in papaya cultivars. In this study, the development of the RKN \u003cem\u003eMeloidogyne javanica\u003c/em\u003e was assessed in the susceptible papaya variety Solo8 over a period of 35 days, using microscopic observation of inoculated roots stained with fuchsin. By 9 days after inoculation (DAI), second-stage juveniles (J2s) were observed migrating through the root cortex and feeding within the vascular cylinder. At 16 DAI, galls containing enlarged J2s had formed within the stele, where feeding sites were established. Young females began appearing within the galls at 26 DAI, and mature pear-shaped females with egg masses were present by 35 DAI. Remarkably, some egg masses were deposited within the cortex, where a new generation of J2s hatched inside the root. Histological cross-sections of galls revealed that feeding sites initiated around the nematode head within the stele by 9 DAI, with four to eight multinucleated giant cells (GCs) present at these sites. These GCs, located within xylem and protophloem elements, were progressively encased by additional cell layers from adjacent vascular and cortical tissues as the nematode developed within the gall at 16, 26, and 35 DAI. The GCs reached maximum size by 16 DAI. Cytoplasmic analysis showed that GCs were rich in proteins, as evidenced by Naphtol Blue Black staining, and that their cell walls were strongly stained for polysaccharides using PAS. This study offers comprehensive histological insights into nematode development within papaya roots, underscoring that screening papaya genotypes for RKN resistance should consider egg mass production within the root tissue.\u003c/p\u003e","manuscriptTitle":"Life cycle of the Root-Knot Nematode Meloidogyne javanica in papaya and histological analyses of root infection and gall development.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-21 04:31:34","doi":"10.21203/rs.3.rs-5377678/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revisions","date":"2025-01-31T12:19:09+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-12-04T18:34:59+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-12-04T11:47:45+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Tropical Plant Pathology","date":"2024-11-09T12:25:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-08T06:50:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Tropical Plant Pathology","date":"2024-11-05T08:56:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"tropical-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tppa","sideBox":"Learn more about [Tropical Plant Pathology](https://www.springer.com/journal/40858)","snPcode":"40858","submissionUrl":"https://www.editorialmanager.com/tppa","title":"Tropical Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0fd96865-cb97-4bff-9561-806ff8e678bb","owner":[],"postedDate":"March 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-03-21T04:31:34+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-21 04:31:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5377678","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5377678","identity":"rs-5377678","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}