The identification, characterization, and management of Rotylenchulus reniformis on Cucumis melo in China

Abstract

The reniform nematode, Rotylenchulus reniformis, is a sedentary root parasite that poses a significant threat to agricultural production in tropical and subtropical regions worldwide. In 2021–2022, a population of R. reniformis was identified in a melon greenhouse in Jimo District, Qingdao, China. To characterize this population, we employed morphological, morphometric, and molecular methods, which confirmed the identity of the nematodes as R. reniformis. Our investigation revealed that R. reniformis successfully infected the roots of melon plants and laid eggs, which could have led to significant crop damage. This report represents the first documented example of R. reniformis infecting melon plants in China. We evaluated several control strategies to combat this nematode, and our results indicated that soil solarization and the use of fosthiazate or chitooligosaccharide copper in combination with soil solarization were effective measures for managing R. reniformis in a greenhouse setting. In addition, combining soil solarization with chitooligosaccharide copper promoted melon plant growth and increased the relative abundance of microorganisms with biocontrol potential.

Background

Melon (Cucumis melo), also known as cantaloupe or muskmelon, is a highly valued crop cultivated in temperate and tropical regions worldwide. China is a leading producer of melons, accounting for approximately 35% of global production, with a yield of 13.87 million tons (FAOSTAT 2020). This fruit is a popular choice for consumers due to its delicious flavor, texture, and taste. Furthermore, several studies have demonstrated that melon is a rich source of health-promoting compounds, including carotenoids, folic acid, polyphenols, vitamins C, and minerals (Maietti et al. 2012; Rodriguez-Perez et al. 2013; Rico et al. 2020). Moreover, melons have potential applications as skin moisturizers and stomachic agents (Ritschel et al. 2004).

Plant-parasitic nematodes (PPNs) are a global problem in the agricultural industry due to the damage they cause to crops and their role in spreading pathogens such as fungi and viruses (Pulavarty et al. 2021; Shao et al. 2023). There are over 4100 species of PPNs described in the literature, and according to surveys of researchers, the reniform nematode (RN) is considered as one of the top 10 PPNs based on its economic impact and scientific importance (Jones et al. 2013). RNs can infect the roots of more than 300 plant species, including cotton, soybean, pineapple, and olive (Robinson et al. 2005; Wubben et al. 2010; Nguyen et al. 2020). Reniform nematodes are mostly found in tropical and warm temperate regions of the world, and they are distributed throughout southern China (Zhang 2010). Infection by R. reniformis results in plant stunting, chlorosis, and yield reduction (Robinson et al. 2005; Nguyen et al. 2020). Moreover, plants infected by R. reniformis often present with disease complexes involving other soil-borne pathogens, such as Fusarium solani, Verticillium spp., and Rhizoctonia solani, leading to even greater losses (Castillo et al. 2010). Compared with most PPNs, R. reniformis is widely adapted to different soil types, including soils with high silt and clay content, and can colonize deep in the soil and maintain an anhydrobiotic state to survive the winter (Robinson 2007). All of these characteristics make it challenging to eliminate this nematode from infested soils. The identification of the reniform nematode species, distribution, and its host range is fundamentally important to effective integrated management.

Various strategies have been developed to manage RNs (Davis et al. 2003; Usovsky et al. 2021), but the most common approach has been the application of chemical nematicides. However, these nematicides are not only expensive but also harmful to the environment and human health (Koenning et al. 2004; Chen et al. 2020). Due to their negative environmental and human health impact, many chemical nematicides like methyl bromide and dibromochloropropane have been banned in agriculture (Noling and Becker 1994; Schneider et al. 2003). To promote healthy and high-quality food production using environmentally friendly, low-cost, and effective methods, integrated pest management (IPM) strategies that combine multiple tactics have been proposed for RN control, such as judicious use of soil solarization and genotype (Chellemi et al. 1993). These sustainable methods can help minimize the use of chemical nematicides while ensuring efficient control of RNs.

In 2021–2022, R. reniformis was detected in the roots of some irregular stunted melon plants (Additional file 1: Figure S1) and rhizosphere soil in the Jimo District of Qingdao, China. The nematode was identified and characterized using a combination of morphological, morphometric, and molecular methods. Furthermore, we investigated the efficacy of soil solarization independently and in combination with fosthiazate or chitooligosaccharide copper treatments to control R. reniformis infection in melon plants under greenhouse conditions. To our knowledge, this is the first report of R. reniformis in C. melo in China, and our findings now lay a foundation for effective management strategies for R. reniformis in greenhouse settings.

Results

Morphological and morphometric characteristics of the nematode

As shown in Table 1, the morphological traits of the R. reniformis population in this study mostly fall within the range of variation in previous studies (Agudelo et al. 2005; Lopez-Nicora et al. 2018; Liu et al. 2023a). The immature female body had a C-shaped form (Fig. 1a), a high and conoid-rounded lip region that blended smoothly with the body contour. It also had a long, well-developed stylet with rounded knobs that slope towards the posterior (Fig. 1b). The vulva was located at the posterior end, between 66.7 and 72.1% of the body length (mean = 71.3%) (Fig. 1a). The body shape of the male was like that of the immature female with a reduced stylet (Fig. 1c, d) and well-developed spicule and gubernaculum (Fig. 1e). The mature female body was kidney-shaped with a well-formed vulva, which had two raised lips and a tail that had a finger-like tip with a prolonged hyaline portion (Fig. 1f).

Table 1 Morphometrics of females and males from the R. reniformis isolate

Fig. 1

The morphological characteristics of R. reniformis from C. melo from a Shandong province greenhouse observed using a compound microscope. Image panels contain pictures of a an immature female, b the anterior part of an immature female, c a male, d the anterior part of a male, e the spicule of a male, and f mature females. st = stylet, v = vulva, sp = spicule. Scale bars = 50 μm

Molecular characterization of the nematode

PCR amplification of the D2–D3 region of the 28S and the ITS1 rRNA gene was performed on the nematode sample described above. Four new sequences of 28S rRNA gene (accession number: OR399163, OR399166, OR404988, and OR404989) and six new sequences of ITS1 in the rRNA gene (OR418490, OR418491, and OR432066–OR432069) were obtained in the present study. The intraspecific variability among the D2–D3 region of the 28S from R. reniformis was found to be considerably high, ranging from 85 to 98%. Sequences of OR399163 and OR399166 (28S rRNA gene) showed similarity values of > 98% with other identified sequences of R. reniformis deposited in the National Center for Biotechnology Information (NCBI) database from the USA (KT003743 and KT003746), and sequences of OR404988 and OR404989 (28S rRNA gene) showed similarity values of > 96% with other identified sequences of R. reniformis deposited in the NCBI database from China (KP054112 and KP054128). Sequences of OR418490, OR418491, and OR432069 (ITS1 rRNA gene) showed similarity values of > 98% with other identified sequences of R. reniformis deposited in the NCBI database from China (GU003948 and KP018567), and sequences of OR432066–OR432068 (ITS1 rRNA gene) showed similarity values of > 98% with other identified sequences of R. reniformis deposited in the NCBI database from China and Japan (GU003962, LC335935, and LC335911).

A phylogenetic tree was constructed using four new D2–D3 sequences in this study and 64 other 28S rRNA gene sequences. R. reniformis is divided into two clades: type A and type B, showing a sister relationship with R. macrodoratus. Two new sequences in this study (OR399163 and OR399166) are clustered in the type A clade, and another two new sequences (OR404988 and OR404989) are clustered in the type B clade (Fig. 2). Similar to the D2–D3 tree, ITS1 in the rRNA genes from R. reniformis population in this study also formed two different subclades. Three new sequences in this study (OR418490, OR418491, and OR432069) are clustered in the type A clade and other three new sequences (OR432066–OR432068) are clustered in the type B clade (Fig. 3).

Fig. 2

A phylogenetic tree based on the alignment of sequences from the D2–D3 region of the 28S rRNA gene of R. reniformis isolated from C. melo and other Rotylenchulus species. The phylogenetic tree was constructed using the maximum likelihood method supported by bootstrap for 1000 replicates. The bootstrap value is given on each node. Four bold marked sequences (OR399163, OR399166, OR404988, and OR404989) were from this study

Fig. 3

A phylogenetic tree based on the alignment of sequences from the ITS1 rRNA gene of R. reniformis isolated from C. melo and other Rotylenchulus species. The phylogenetic tree was constructed using the maximum likelihood method supported by bootstrap for 1000 replicates. The bootstrap value is given on each node. Six bold marked sequences (OR418490, OR418491, and OR432066–OR432069) were from this study

Assessing melon plant roots for infection by the isolated R. reniformis

We inoculated the roots of melon plants with the newly hatched J2s of R. reniformis and observed successful penetration at 4 days post-inoculation (dpi). Females protruding from the roots appeared swollen at 7 dpi, and their bodies became kidney-shaped by 10 dpi. The females laid eggs at 15 dpi (Additional file 1: Figure S2). Then, the egg masses were hand-picked from the roots and placed in Petri dishes containing sterilized water. Hatched J2s of R. reniformis were detected at 3 days after incubation. The results verified that our isolate of R. reniformis could successfully infect the roots of melon plants and complete its life cycle within 18 days at a temperature of 26 ± 3°C.

Population dynamics of the R. reniformis and its response to common control strategies in the greenhouse

The greenhouse was highly infested with R. reniformis with an original average population density of 574.2 ± 15.3 and 345.7 ± 18.4 nematodes/100 g of soil in 2021 and 2022, respectively. In 2021, after the transplanting of melon plants in greenhouse plots, the infection of R. reniformis was monitored in the root system. The number of infected R. reniformis peaked 8 days after transplanting (October 2^nd), with an average nematode density of 1140.3 ± 89.2 nematodes/10 g of roots. The nematode density then began to decline before increasing again and peaking at 93.0 ± 5.9 nematodes/10 g of roots at 23 days after transplanting (October 17^th) (Fig. 4a). The density of R. reniformis in the rhizosphere gradually increased from September to December when it reached a peak density of 2269.3 ± 72.3 nematodes/100 g of soil nearly three months after transplanting (December 14^th) (Fig. 4b). A similar population dynamics of the R. reniformis in melon plants roots and rhizosphere was detected in 2022 (Fig. 4c, d).

Fig. 4

Population dynamics of R. reniformis and treatment efficacy in greenhouse experiments in the years 2021 and 2022. a The population density of R. reniformis on C. melo roots under different treatments in 2021. b The population density of R. reniformis in the rhizosphere soil of C. melo under different treatments in 2021. c The population density of R. reniformis on C. melo roots under different treatments in 2022. d The population density of R. reniformis in the rhizosphere soil of C. melo under different treatments in 2022. The C. melo plants were transplanted into infested soil on September 24^th, 2021 and October 2^nd, 2022. The number of R. reniformis in C. melo roots and rhizosphere soil was recorded 3 days after the transplanting of C. melo seedlings. S: soil solarization; F: fosthiazate; SF: combination of soil solarization and fosthiazate; SC: combination of soil solarization and chitooligosaccharide copper; BC: blank control; dat: days after transplanting

We then tested the efficacy of different potentially nematicidal treatments against R. reniformis. Soil solarization (S), fosthiazate (F), a combination of soil solarization and fosthiazate (SF), or a combination of soil solarization and chitooligosaccharide copper (SC) were all used as treatments against R. reniformis in greenhouse assays. All four treatments resulted in reduced nematode infection in melon plant roots and the rhizosphere compared with the blank control (BC) in 2021 and 2022 (Fig. 4). The SF and SC treatments led to significantly reduced R. reniformis infection compared to the F and BC treatments (P < 0.05). In 2021, the S and F treatments resulted in 98.1% and 78.2% reductions in nematode density, respectively, compared to the BC treatment when root samples were taken 8 days after transplanting (October 2^nd). The SF and SC treatments were the most effective and nearly eliminated nematodes from melon plant roots 8 days after transplanting (October 2^nd) (Fig. 4a). The F treatment initially reduced R. reniformis in the rhizosphere soil two weeks after transplanting, but this effect reversed as nematodes proliferated during the middle and later part of the growing season (Fig. 4b). In 2022, compared with the BC treatment when root samples were taken 10 days after transplanting (October 12^th), the S, F, SF, and SC treatments decreased nematodes in the infected roots by 98.2%, 91.3%, 100%, and 100%, respectively. The melon plants treated with S, particularly the combinations of SF and SC, had fewer R. reniformis in the rhizosphere soil throughout the growing season compared to the other treatments (Fig. 4b, d).

We also measured plant growth parameters (shoot weight and shoot length) of melon plants under different treatment conditions. We found that the SC treatment was the most effective in promoting melon plant growth. In 2021, the shoot weight was approximately twice as high as the BC, whereas the shoot length was 60% higher than the BC. We also detected a significant increase in the shoot weight and length after SC treatment in 2022 (Additional file 2: Table S1).

The effects of S and SC treatments on the rhizosphere microbiota

Melon plants treated with S clearly (Fig. 4) had fewer R. reniformis nematodes in their roots than plants treated with F. The combination treatment of SC, which involved the use of S and chitooligosaccharide copper, proved effective in controlling R. reniformis and contributed to improved plant growth. We next evaluated the effect of S and SC treatments on the rhizosphere microbiota by measuring the relative abundance of soil microbial communities at the genus level. Unidentified Gemmatimonadaceae, Vicinamibacterales, Vicinamibacteraceae, and Sphingomonas were the dominant bacteria in all rhizosphere soils (Fig. 5a and Additional file 2: Table S2). The three most abundant fungi were from unidentified Basidiomycota, Xeromyces, and Cladosporium (Fig. 5b and Additional file 2: Table S3). To determine the alpha diversity of rhizosphere microbiota, the Shannon index (reflecting species richness) and abundance-based richness estimation (ACE, reflecting species abundance) indexes were calculated. The results showed that the Shannon index of bacteria in S and SC was significantly lower than that in BC, but there was no significant difference between the ACE indexes of bacteria in the rhizosphere treated with SC and BC. However, a pronounced decline in the ACE index was observed with the S treatment. Both the S and SC treatments significantly increased the Shannon index of fungi, whereas no significant differences were observed in the ACE index between S, SC, and BC treatments. A principal coordinate analysis (PCoA) showed significant differences in the structure of bacterial and fungal communities in the rhizosphere treated with S, SC, and BC (Additional file 1: Figure S3).

Fig. 5

The relative abundance of rhizosphere microbiota in the rhizosphere soil of C. melo. The relative abundance of a bacteria, and b fungi at the genus level

The Gram-staining phenotypes of microbiota were predicted by BugBase (Fig. 6a, b). An integrated ANOVA analysis was conducted to examine the relative abundance of Bacillus spp. within the gram-positive bacteria. The results indicated that soils treated with SC had a higher relative abundance of Bacillus spp. compared to those treated with BC (Fig. 6c). We also used high-dimensional biomarkers to group fungal communities at the genus level in the rhizosphere treated with S, SC, and BC by ANOVA analysis. The relative abundance of the 18 most abundant genera of fungi in rhizosphere treated with S, SC, and BC is shown in Fig. 7. Certain fungi with potential biocontrol activity, such as Penicillium, Polypaecilum, Gibellulopsis, Wickerhamomyces, and Hannaella, were significantly more abundant in the rhizosphere treated with S and SC (Fig. 7). Inversely, the abundance of potentially plant pathogenic fungi, such as Fusarium and Metacordyceps, were significantly less abundant in the rhizosphere treated with S and SC (Fig. 7).

Fig. 6

Microbial community phenotypes and bacterial community composition in the rhizosphere soil of C. melo based on BugBase prediction. a, b Classification of bacteria based on Gram staining. c The relative abundance of Bacillus spp. in the rhizosphere of soils treated with BC, S, or SC. Each bar represents the mean ± SD. The different lowercase letters on the bars indicate significant differences according to one-way ANOVA (P < 0.05, Tukey’s HSD test). BC: blank control; S: soil solarization; SC: combination of soil solarization and chitooligosaccharide copper

Fig. 7

The relative abundance of the 18 most abundant genera of fungi in the rhizosphere soil of C. melo treated with S, SC, or BC. The red solid circles denote fungi with plant disease biocontrol potential. The green solid circles denote fungi with plant pathogenic potential. Each bar represents the mean ± SD. The different lowercase letters on the bars indicate significant differences according to one-way ANOVA (P < 0.05, Tukey’s HSD test). S: soil solarization; SC: combination of soil solarization and chitooligosaccharide copper; BC: blank control

Discussion

R. reniformis is a parasitic nematode that can infect a wide range of crops and vegetables, leading to severe damage and significant yield losses in agriculture. In this study, we first report the occurrence of R. reniformis on melon plants in China and provide detailed morphological and molecular characterizations of this species. Moreover, we also assessed the population dynamics of R. reniformis in the greenhouse and evaluated the efficacy of several control strategies.

There are several tools available to identify different species of Rotylenchulus. However, distinguishing between species based on their physical characteristics is not very effective due to the high variability of some features in Rotylenchulus. Ribosomal RNA genes have been used effectively in molecular diagnostics to uncover the diversity within the Rotylenchulus genus (Van Den Berg et al. 2016; Palomares-Rius et al. 2018). Intraspecific and intra-individual rRNA variation is commonly found in the genus Rotylenchulus. There is evidence that higher rRNA polymorphisms are associated with increased growth and reproduction (Elser et al. 2000), and Qing et al. (2020) suggested that the rRNA copy number in R. reniformis may still be expanding. In the present study, we found two distinct types of 28S and ITS from R. reniformis, which is consistent with the findings of previous research by Van Den Berg et al. (2016) and Palomares-Rius et al. (2018).

In China, R. reniformis has been reported in the southern regions of Shanghai, Guangdong, Guangxi, Hainan, Anhui, Sichuan, Fujian, Guizhou, Jiangxi, Hubei, Zhejiang, and Yunnan (Zhang 2010; Liu et al. 2023a), posing a serious threat to the production of cotton, banana, citrus fruits, and vegetables in those areas. To our knowledge, this is the first report of R. reniformis on melon plants in China. In other part of the world, we only found reports about the infection of R. reniformis in melon plants in Brazil, which caused high yield losses of melon when there were high population densities of the reniform nematode in the field (Moura et al. 2002). Gustavo et al. (2005) evaluated the resistance of eight cucurbit genotypes to R. reniformis under greenhouse conditions and found that R. reniformis presented high reproduction in two tested C. melon varieties. Similarly, our study showed that R. reniformis can reproduce in the roots of the melon plants and complete its life cycle within 18 days, which is similar to that reported for R. reniformis on susceptible soybean (19 days) (Rebois 1973) and cotton (17 days) (Robinson 2007). In some areas of the USA, the reniform nematodes caused greater loss than root-knot nematodes due to the shorter life cycle and higher reproductive rate (Robinson 2007). The potential damage of R. reniformis necessitates strict testing to detect R. reniformis before seedling transportation, especially in greenhouse planting areas of China, to avoid great losses in vegetable production.

There is also an urgent need to determine the efficacy of environmentally friendly control methods to prevent the spread of R. reniformis and ensure the sustainable development of the vegetable industry. We evaluated the ability of S, F, SF, and SC treatments to control R. reniformis in the greenhouse. Chitooligosaccharide is the degradation product of chitosan, the second most abundant polymer in nature after cellulose (Fan et al. 2023). Previous studies have reported that chitooligosaccharide has antifungal, antibacterial, and plant growth promotion properties and can enhance plant disease resistance (Guan and Feng 2022; Liu et al. 2023b). Chitooligosaccharide and its modified derivatives have been widely used in agriculture due to their excellent biocompatibility and low cost (Fan et al. 2022; Liu et al. 2023b). In this study, we found that soil solarization had a significant effect on the population of R. reniformis, which was consistent with a previous report (Chellemi et al. 1993). Moreover, combining soil solarization with fosthiazate or chitooligosaccharide copper had the greatest efficacy in eliminating nematodes from melon plant roots and rhizosphere soil. Additionally, the application of chitooligosaccharide copper after soil solarization can significantly improve melon plant growth.

Many studies have demonstrated that soil microbial communities influence plant growth and disease resistance under biotic and abiotic stress. Our microbiome analysis showed that treating soil with SC significantly increased the abundance of Bacillus. Bacillus species such as B. cereus, B. subtilis, B. megaterium, and B. velezensis have been previously shown to provide effective control against PPNs in field experiments, with some species even promoting plant growth (Cao et al. 2019; Yin et al. 2021; Zhou et al. 2021; Tian et al. 2022). For example, the B. velezensis strain Bv-DS1 isolated from tidal soil can suppress M. incognita infection in tomato by inducing local resistance and increasing the fresh weight of tomato by producing IAA (Hu et al. 2022). We also evaluated differences in the soil fungal community compositions in soils treated with S, SC, and BC. The results suggest that S and SC treatments increase the abundance of some fungal species with plant disease biocontrol potential.

Conclusions

In this study, we identified and characterized the occurrence of R. reniformis on melon plants in China and compared the efficacy of several control strategies in the greenhouse. The findings of our study offer valuable insights into the potential damage caused by this nematode and provide useful information on effective control methods.

Methods

Plant and soil samples

In 2021–2022, some irregular stunted melon plants were observed in a greenhouse in the Jimo District (36.37°N; 120.51°E) of Qingdao, China. Roots of melon plants and rhizosphere soil were sampled and taken back to the Nematology Laboratory of Qingdao Agricultural University for identification.

The collection and observation of nematodes

Nematodes were extracted from the rhizosphere soil of melon plants using the Baermann tray method with some modifications (Barker 1985). The characteristics of immature females and males were observed and photographed by a compound microscope equipped with a Nomarski differential interference contrast (Axioscope 5, Zeiss, Germany). Measurements were performed using a drawing tube attached to the microscope. The females in the root tissue were stained using the sodium hypochlorite-acid fuchsin method (Byrd et al. 1983). Briefly, infected roots were placed in a beaker containing 50 mL of 1.5% NaOCl for 5 min, followed by washing with sterile water until all residual NaOCl had been removed. One milliliter of acid fuhsin was added to the beaker and incubated at 100°C in a water bath for 30 s. After washing with sterile water, the roots were placed in a beaker containing 20 mL of acid glycerol and boiled. Roots were then dissected, and the female nematodes were observed and photographed by a compound microscope (Axioscope 5, Zeiss, Germany).

Molecular characterization

DNA extraction and PCR

Genomic DNA was extracted from individual immature females according to previously described protocols with some modifications (Castagnone-Sereno et al. 1995; Holterman et al. 2006). In summary, a single nematode was transferred to a 0.2 mL PCR tube containing 10 µL of lysis buffer (60 mg/mL proteinase K, 40 mM NaCl, and 20 mM Tris-HCl at a pH of 8.0). Samples were incubated at 65°C for 90 min, followed by 95 °C for 10 min and then stored at − 20°C for later use.

The D2–D3 region of the 28S rRNA gene and the ITS1 rRNA gene were amplified using the primer pairs D2A/D3B and TW81/AB28 (Additional file 2: Table S4). A total reaction volume of 25 µL was used for PCR. Each reaction contained 12.5 µL of I-5™ 2 × High-Fidelity Master Mix (TSINGKE, China), 2 µL of template DNA, 2 µL of forward and reverse primers (10 µM), and 8.5 μL of nuclease-free water. The PCR cycling conditions were as follows: 98°C for 3 min (initial denature) followed by 30 cycles of 98°C for 10 s (denature), 55°C for 10 s (primer annealing), 72°C for 15 s (extension), and a final extension at 72°C for 5 min.

Sequencing and phylogenetic analyses

PCR products were purified using the E.Z.N.A. Gel Extraction Kit (Omega, USA) according to the manufacturer's instructions. Amplified fragments were cloned into the pClone007 Blunt Vector (TSINGKE, China) according to the manufacturer's instructions and sequenced in two primer directions by TSINGKE Biological Technology (Qingdao, China). The sequences obtained from the R. reniformis isolates were submitted to GenBank. Phylogenetic tree analyses were performed with MEGA7 software using the maximum likelihood method of 1000 replicates (Kumar et al. 2016).

Assessing the pathogenicity of R. reniformis

Egg masses of R. reniformis were hand-picked and placed in a petri dish. Eggs were then washed with 0.5% NaOCl for 1 min, followed immediately by five washes with sterile water. The egg masses were re-suspended in sterile water and incubated at 26°C for hatching. After 3 days, the newly hatched J2s were collected for melon plants inoculations. Melon seeds were surface sterilized with 1% NaOCl and germinated on moist filter paper. After 3 days, the germinated melon seeds were transferred to individual pots (9 cm × 9 cm × 10 cm) containing sterile substrate (70% peat and 30% vermiculite). Plants were cultivated at 26 ± 3°C. When melon plants reached the two-leaf stage, we used a hole puncher to punch three small holes at a depth of 1 cm in the soil surrounding the root zone of each melon plant, and each hole was inoculated with 300 R. reniformis J2s in 1 mL of sterilized water, and then carefully covered with sterile substrate. Three days post-inoculation, the roots of melon plants were stained using acid fuchsin and observed under a stereoscope. Three independent experiments were carried out with similar results.

Greenhouse trial design

A greenhouse trial was carried out in Jimo in a greenhouse where we found the R. reniformis. Two greenhouse experiments were conducted from September to December 2021 and October to December 2022. The R. reniformis population densities in the greenhouse were detected before conducting the greenhouse experiments. The rhizosphere soil of each plot was sampled at five points, and 100 g of mixed soil from each point was used for nematode collection according to the Baermann tray method. R. reniformis was counted under a microscope (Olympus, Japan). Five treatments were applied: solarization [soil covered with high-density polyethylene film (0.2 mm; Shandong Xinyu Engineering Material Company Ltd.) during the whole month of August], fosthiazate (Fuqiduo; 10% fosthiazate granules; 80 g/plot), solarization followed by fosthiazate, solarization followed by chitooligosaccharide copper (Qingdao Zhongke Xinghai Biotechnology Co., Ltd; 5% chitooligosaccharide copper, 2.7% chelated copper; 200 mL/plot), and water (blank control). All treatments were arranged in a randomized complete blank design, and each treatment was applied to three replicated plots. Melon seedlings at the two-leaf stage were then transplanted in each plot. The area of each plot was 30 m², with four rows in each plot and 30 melon plants per row. The number of R. reniformis in the roots of melon plants and rhizosphere soil was recorded 3 days after the transplanting of melon seedlings. 28 days after the transplanting, five melon plants were randomly sampled from each plot (a total of 15 plants of each treatment), and growth parameters (shoot weight and shoot length) of melon plants were recorded. The temperature of the greenhouse was about 25–34°C. Thirty days after transplanting, the rhizosphere soil was collected for Illumina NovaSeq sequencing.

DNA extraction, Illumina NovaSeq sequencing, and analysis

An E.Z.N.A. Soil DNA Kit (Omega, USA) was used to extract DNA from rhizosphere samples according to the manufacturer’s instructions. The DNA quantity and quality were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) and electrophoresis on a 1% agarose gel. The V3–V4 region of bacterial 16S rRNA and the ITS1 region of the fungal internally transcribed spacer were amplified using primer pairs 338F/806R and ITS1F/ITS2R (Additional file 2: Table S4). PCR products were purified using the EasyPure® Quick Gel Extraction Kit (TransGen, China) and sequenced on the Illumina Novaseq 6000 platform (Biomarker Technologies, China).

The bioinformatics analysis was performed using BMKCloud (http://www.biocloud.net/). Briefly, raw paired-end sequence reads were primarily filtered using Trimmomatic v0.33 (Bolger et al. 2014) and primer sequences were identified and removed using Cutadapt (Martin 2011). QIIME2 (Bolyen et al. 2019) and DADA2 (Callahan et al. 2016) were used to remove chimeric and short reads. Sequences with > 97% sequence similarity were clustered to one operational taxonomic units (OTUs) by USEARCH 10.0 (Rognes et al. 2016). For the microbial community analysis, alpha diversity indices, such as the Shannon index and ACE index (Lee et al. 1992; Spellerberg et al. 2003) were calculated using the picante database (Kembel et al. 2010). A beta diversity analysis was performed using qiime software (http://qiime.org/). A principal coordinates analysis (PCoA) (Gower 1966) was performed using R software (v4.1.2; http://www.r-project.org/). Significant differences in community structures among groups were assessed using an ANOVA followed by the Tukey’s honest significant difference (HSD) tests. The phenotypic traits of microbial communities were predicted by BugBase (http://bugbase.cs.umn.edu).

Statistical analysis

Data derived from greenhouse experiments were analyzed for significance by one-way ANOVA followed by Duncan’s multiple range test (P ≤ 0.05) using the software SPSS (version 16.0 for Windows).