Introduction
The rice root-knot nematode, Meloidogyne graminicola, Golden and Birchfield, is one of the most damaging nematode species. It has emerged as a serious threat to rice production due to its worldwide distribution, particularly in Southeast Asia, which constitutes 90% of total rice production (Rusinque et al., Reference Rusinque, Maleita, Abrantes, Palomares-Rius and Inácio2021). This endoparasitic sedentary nematode is prevalent in almost all rice cropping systems, including lowland, upland, irrigated and deepwater (Pokharel et al., Reference Pokharel, Abawi, Duxbury, Smat, Wang and Brito2010; Mantelin et al., Reference Mantelin, Bellafiore and Kyndt2017). It causes considerable damage to rice root systems in nurseries and significant yield loss in the field ranging from 70 to 80% in lowland and upland rice cultivation (Patil and Gaur, Reference Patil and Gaur2014; Dimkpa et al., Reference Dimkpa, Lahari, Shrestha, Douglas, Gheysen and Price2016; Galeng-Lawilao et al., Reference Galeng-Lawilao, Kumar and Waele2018).
Rice RKN infective second-stage juveniles (J2) penetrate the root system and develop nematode-specific feeding sites. These feeding sites are multinucleated giant cells (GC) formed through endomitosis and cellular hypertrophy and are often referred to as ‘metabolic sinks’ due to the diversion of host nutrition to the nematode. Further, terminal hook-shaped galls are formed on roots due to the division of the pericycle cells which are also the direct representation of nematode infestation and susceptibility of rice plants to RKN (Ralmi et al., Reference Ralmi, Khandaker and Mat2016). Among the physiological processes that have often been considered responsible for the reduction of the photosynthetic activity in nematode-infested plants are decreased chlorophyll content (Nagesh and Dhawan, Reference Nagesh and Dhawan1988), changes in stomatic conductance (Saeed et al., Reference Saeed, MacGuidwin and Rouse1998) and photochemical limitations (Schans and Arntzen, Reference Schans and Arntzen1991). This results in a lower photosynthetic rate and ultimately the lower translocation of photosynthates lead to stunting, chlorosis, loss of vigour and yield losses (Swain and Prasad, Reference Swain and Prasad1988; Wenting and Deliang, Reference Wenting and Deliang2007; Chen et al., Reference Chen, Sikandar, Ahmad, Luo and Wu2022).
M. graminicola is well-adapted to the wide variety of agro-ecosystems in which rice is cultivated. Today, the impact of water scarcity has shifted focus to water-saving rice cultivation systems such as aerobic rice. Consequently, the agronomic practices in aerobic, non-puddling and non-flooding or non-saturated fields conditions have led to an increase in nematode infestations (Ravindra et al., Reference Ravindra, Sehgal, Narasimhamurthy, Krishnappa and Imran2017). The options to control M. graminicola are limited. De-registration of frontline nematicides and their hazardous impact on health and the environment has shifted the focus to other management strategies (De-Waele et al., Reference De-Waele, Das, Zhao, Tiwari, Shrivastava, VeraCruz and Kumar2013; Dimkpa et al., Reference Dimkpa, Lahari, Shrestha, Douglas, Gheysen and Price2016). The low-cost, environmentally beneficial and sustainable approach is the use of resistant cultivars. Most of the Asian rice germplasm screened so far is susceptible to RKN, and only a few of them were found to be resistant (Dimkpa et al., Reference Dimkpa, Lahari, Shrestha, Douglas, Gheysen and Price2016; Subudhi et al., Reference Subudhi, Dash, Meher, Mohapatra and Mishra2017). Therefore, it is a pressing need to identify nematode resistant sources from wide range of germplasm which could be deployed in breeding programs for its management. Natural plant resistance to RKN had already been reported from O. glaberrima and O. longistaminata (Soriano et al., Reference Soriano, Espiritu, Schmit, Brar and Reversat1999) through pot experiments. One accession (WL02) of O. longistaminata and three accessions of O. glaberrima (TOG7235, TOG5674 and TOG5675) were resistant to RKN (Soriano et al., Reference Soriano, Espiritu, Schmit, Brar and Reversat1999). As the study was limited to only three accessions of O. glaberrima, so there is need to explore more accessions for resistance. Despite the identification of O. glaberrima (West Africa cultivar) as resistant source for RKN, it is not economically important due to low yielding in comparison to Asian rice cultivars. Therefore, it is essential to introgress RKN resistance from West African cultivars into economically important and high yielding Asian rice cultivars through backcross breeding. Introgression of RKN resistance into Asian cultivars has been initiated but was not successful so far, because progenies from interspecific crosses did not express same resistance level as that of O. glaberrima (Plowright et al., Reference Plowright, Coyne, Nash and Jones1999). Sexual incompatibility and hybrid sterility further limits the efforts of combining M. graminicola resistance and yielding potential from these two different rice species. However, fertility can be achieved through continual backcrossing for fewer generations (Jones et al., Reference Jones, Dingkuhn, Aluko and Semon1997, Cabasan et al., Reference Cabasan, Kumar and De Waele2018).
Besides the phenotypic evaluations, an insight into the genetic analysis or molecular approaches enables us to ascertain the number of genes involved in resistance. It has been documented that resistance to rice RKN is not monogenic but multigenic in nature (Prasad et al., Reference Prasad, Vijayakumar, Sankar, Varaprasad, Srinivasa and Kondala2006; Dimkpa et al., Reference Dimkpa, Lahari, Shrestha, Douglas, Gheysen and Price2016; Galeng-Lawilao et al., Reference Galeng-Lawilao, Kumar and Waele2018). Several natural host resistance genes and proteinase/protease inhibitor genes have been associated with nematode resistance, and other plant species could share similar resistance genes (Ali et al., Reference Ali, Azeem, Li and Bohlmann2017). The availability of whole-genome rice sequence information will lead to identifying the ortholog sequences known from other crop systems like cowpea, potato, maize and tomato for nematode host resistance genes (Hepher and Atkinson, Reference Hepher and Atkinson1992; Paal et al., Reference Paal, Henselewski, Muth, Meksem, Menéndez, Salamini, Ballvora and Gebhardt2004; Roderick et al., Reference Roderick, Tripathi, Babirye, Wang, Tripathi, Urwin and Atkinson2012; El-Sappah et al., Reference El-Sappah, Islam, El-awady, Yan, Qi, Liu, Cheng and Liang2019). It will provide information about a gene sequence's structure and function that could be associated with nematode resistance. The identification of genetic variation in terms of SNPs/InDels through in silico approach could be used for functional validation. In plants, the preponderance of traits of interest is linked with SNPs and thought to bring individual variation, community diversity and the evolution of species (Shirasawa et al., Reference Shirasawa, Bertioli, Varshney, Moretzsohn and Leaf-Bertioli2013). Thus, the present study was conceded to (i) screen potential sources of RKN resistance among O. sativa genotypes and O. glaberrima accessions and (ii) study histological variations between resistant and susceptible genotypes, along with in silico studies using bioinformatics tools to identify SNPs/InDels for resistance.
Materials and methods
Experimental materials
Eighty-three genotypes of O. sativa and forty-two accessions of O. glaberrima were evaluated along with three susceptible checks (Pusa1121, PR116 and PR121) in nematode infested soil during the summer seasons of 2017 and 2018. Also, all the genotypes were planted in non-infested soil to measure plant growth parameters. The O. glaberrima accessions were procured from International Rice Research Institute, Manila, Philippines, and maintained at the School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana. All 128 genotypes were raised in a randomized complete block design in three replications. Each genotype was planted in triplicate with a plant to plant distance of 15 cm and 50 cm from row to row. Standard agronomical practices were followed for raising the crop.
Preparation of RKN infested plot
The nematode infested plot was established during 2015–16 using roots of RKN infested rice seedlings, and soil collected from fields of Punjab Agricultural University, Ludhiana as well as different regions of Punjab (Ludhiana, Faridkot and Sangrur). The population of M. graminicola from the Punjab region used in the experiment was previously identified and the identity of the nematode was confirmed by the method described by Yik and Birchfield (Reference Yik and Birchfield1979). A raised bed was prepared with nematode infested soil and infested seedlings which were directly transplanted on the bed for rapid spreading of M. graminicola infestation uniformly throughout the plot. After infested seedlings reached maturity, shoots were separated from roots. Infested roots were chopped into small pieces and mixed in the upper layer of soil. The plot was kept saturated but not flooded to maximize the reproduction or RKN. During the growth period of seedlings, RKN completes their life cycles and helps in increasing the soil nematode population density. Later on, the nematode population density was maintained throughout the year by sowing PR121 during the summer season and an alternative host (wheat) in the winter season.
Experimental design and assessment of nematode population in soil
M. graminicola infested plot (30 × 8 m) was sub-divided into ten micro plots, and the size of each micro plot consisted of 3 × 5 m (Online Supplementary Fig. S1). The micro plots were separated from each other by 1 m. An earthen levee was constructed around infested plot to prevent the spread of nematode to a non-infested plot, which was maintained at a buffer space of 10 m from the infested plot. Six composite soil samples from each microplot were taken to determine the initial soil nematode population density. The soil nematode population was determined using Cobb's sieving and decanting method (Cobb, Reference Cobb1918). The average initial population density of 10 different micro plots was assured uniform throughout the nematode infested plot by a continuous infestation of nematode infested soil/root samples in the infested plot before initiating the screening experiment.
Screening and rating
All genotypes were raised in nursery beds and transplanted after 25 days of sowing in the nematode infested plot with an average initial population density of 1 J2/cc soil. The nematode infested plot was not flooded but kept saturated during the experiment to maximize the penetration of juveniles inside the roots. Standard agronomical practices were followed for the growth of seedlings. Nine plants of each entry from three replications were uprooted carefully after sixty days of infestation, and roots were washed immediately to count the number of galls per total root system. The incidence of root-knot disease was measured by a root gall index scale of 1–5 (Gaur et al., Reference Gaur, Singh, Kumar, Kumar and Singh2001). The rating was done 1 for 0–1 gall (highly resistant), 2 for 1–10 galls (resistant), 3 for 11–30 galls (moderately resistant), 4 for 30–100 galls (susceptible) and 5 for >100 galls (highly susceptible). The final soil nematode population density was assessed from 250 cc soil collected with a soil auger at a depth of 35 cm of three plants of each genotype from three replications. The reproduction factor (Rf) was calculated by dividing the final population density (Pf) by the initial population density (Pi) of juveniles.
Effect of nematode infestation on plant growth parameters
Data on different growth performance factors like plant height (cm) and root length (cm) from both uninfected and nematode infested plots were recorded among six O. glaberrima resistant accessions and susceptible checks (Pusa1121, PR116 and PR121) in three replicates over two years. The height of each plant was measured from the ground surface to the tip of the longest leaf. Root length was measured from the ground surface to the longest root.
Histopathological studies
The nematode infested roots of susceptible and resistant plants were stained with acid fuschin and were observed under a stereoscope microscope to check the presence of nematode/egg masses inside the gall. Subsequently, M. graminicola infested roots of PR121 and O. glaberrima acc. IRGC102196 was collected after 30 days of transplanting. The roots were first gently washed in running tap water to remove adhered soil. Root galls were hand sectioned by placing samples in potato pith. The samples were then mounted on DPX with a coverslip. The slides were observed under a microscope equipped with a digital camera and computer imaging system using software NIS Elements F 30 at 4X and 10X. 4-5 root segments (1–2 cm long) of both genotypes were selected and excised for scanning electron microscopic analysis. The cross and longitudinal sections (4–5 μm long) were further cut from root segments and fixed in 2.5% (v/v) glutaraldehyde at 40°C overnight. After draining the fixative, the samples were treated with 0.1 M CaCO3 buffer with an interval of 15 min each three times. Later on, samples were treated with 1% Osmium tetroxide (OsO4) for 1–2 h at 40°C. Again three washings were given with 0.1 M CaCO3 after draining OsO4. The samples were then dehydrated in ethanol concentrations from 30% to 100% v/v (30, 50, 70, 80, 90 and 100%). The samples were later dried at a critical point and placed in vacuum desiccators overnight. A coating of carbon on each sample was done with the help of a sputter coater; these samples were then observed under a scanning electron microscope (SEM).
In silico analysis of nematode resistance genes
A total of 14 cloned nematode resistance genes from different crops were selected (Online Supplementary Table S1) to identify the orthologs in O. glaberrima acc. IRGC102206. The sequences of nematode resistance genes were retrieved through the NCBI database (www.ncbi.nlm.nih.gov). The FASTA sequence files were BLAST against Oryza sativa Nipponbare reference genome of rice on RGAP (Rice Genome Annotation Project) (http://rice.plantbiology.msu.edu/analyses_search_blast.shtml). Genome sequences of the O. sativa indica group and O. glaberrima were retrieved from Ensembl Plants (https://plants.ensembl.org/species) for comparative analysis. The nucleotide sequences of Oryzacystatin-1 and Oryzacystatin-2 genes were retrieved from whole-genome resequencing data of O. sativa cv. PR121 and O. glaberrima acc. IRGC102206 (our unpublished data) to confirm sequence variations. Both genes were aligned separately between PR121 and O. glaberrima acc. IRGC102206 through CLUSTAL software using the CodonCode Aligner tool (https://www.codoncode.com/).
Statistical analysis
The average initial nematode population density from each microplot, final soil nematode population density, reproduction factor, root gall index and growth parameters were analysed through analysis of variance using a generalized linear model (GLM) of SAS software version 9.4 (SAS Institute, Cary, NC). The average root gall index of each genotype was compared using Tukey's host significant difference (Tukey's HSD) test.
Results
Host response to M. graminicola
The initial nematode population density was an average of 1 J2/gram of soil throughout the nematode infested plot. There was no significant difference in the initial soil nematode population density (F9, 47 = 1.04; P > 0.426) among the micro plots. On confirmation of the uniform distribution of nematode population density in the infested plot, the screening of rice germplasm was done. The germplasm screened for their response to RKN in the present studies were categorized into three groups viz. O. sativa basmati genotypes, O. sativa non-basmati genotypes and O. glaberrima accessions. These three groups showed significant variations in their susceptibility to M. graminicola. Amongst the three groups, root gall numbers and final soil nematode population were higher in O. sativa genotypes than in O. glaberrima accessions. Fifteen basmati genotypes were highly susceptible, while 13 were rated as susceptible and five as moderately susceptible based on the root gall index (Table 1). Similarly, forty-one and nine non-basmati genotypes were classified as highly susceptible and susceptible, respectively (Table 2). The reproduction factor in all O. sativa genotypes was >1.5, indicating that these genotypes reproduced nematode populations. Root gall index was also >3 in all the susceptible genotypes. Root galling was found throughout the root system along with characteristic terminal hook-shaped galls at the root apex as observed in susceptible control PR121 (Fig. 1a).
Table 1. Average soil nematode population, reproduction factor (Rf), root gall index and the response of O. sativa genotypes (Basmati) for the consecutive two years against M. graminicola
Different lowercase letters indicate significant differences among basmati genotypes as determined by Tukey's honest significant difference (Tukey's HSD) test (P < 0.05). HS, S and MS stand for highly susceptible, susceptible and moderately susceptible, respectively.
Table 2. Average soil nematode population, reproduction factor (Rf), root gall index and the response of O. sativa genotypes (non-basmati) for the consecutive two years against M. graminicola
Different lowercase letters indicate significant differences among non-basmati genotypes as determined by Tukey's honest significant difference (Tukey's HSD) test (P < 0.05). HS and S stand for highly susceptible and susceptible, respectively.
Figure 1. Root-knot nematode infested rice plants showing typical galls on root system of susceptible check O. sativa cv. PR121 (a) O. glaberrima acc. IRGC102196 (b) and O. glaberrima acc. IRGC100983 (c).
O. glaberrima accessions exhibited very few galls (Figs 1b and 1c), and the reproduction factor was <1, indicating that they did not favour the build-up of nematode populations. Three O. glaberrima accessions (IRGC102196, IRGC102538 and IRGC102557) were highly resistant and promising for utilization in nematode resistance breeding. Thirty-three and six O. glaberrima accessions were resistant and moderately resistant, respectively (Table 3). Statistical significant differences were observed for final soil nematode population density, reproduction factor and root gall index among the genotypes (Online Supplementary Table S2). The resistant accessions were also evaluated in the pots filled with nematode infested soil @ 1 J2 per cc soil. The reactions of the O. glaberrima accessions were similar to those in the nematode infested plot (data not presented).
Table 3. Average soil nematode population, reproduction factor (Rf), root gall index and the response of O. glaberrima accessions for the consecutive two years against M. graminicola
Different lowercase letters indicate significant differences among Oryza glaberrima accessions as determined by Tukey's honest significant difference (Tukey's HSD) test (P < 0.05). HR, R, MR and S stand for highly resistant, resistant, moderately resistant and susceptible, respectively.
Staining and teasing the mature gall of susceptible check PR121 showed adult female, egg masses (Online Supplementary Fig. S2a) and juveniles released from root galls (Online Supplementary Fig. S2b). Only a few juveniles penetrated O. glaberrima, and no egg masses were seen inside the roots after 60 days of nematode infestation (Online Supplementary Fig. S2d). However, many juveniles were observed in the roots of O. sativa cv. PR121 (Online Supplementary Fig. S2c). The average soil nematode population per 250 cc soil and root gall index among susceptible checks, highly resistant, and resistant accessions of O. glaberrima are presented in Online Supplementary Fig. S3. Soil nematode population per 250 cc soil was higher in susceptible checks (⩾500) compared to O. glaberrima accessions. The soil nematode population was multiplied by >2 fold in susceptibles against <1 fold in O. glaberrima accessions.
Effect of M. graminicola on growth parameters
The present studies revealed that infestations of M. graminicola had a significant suppressive effect on plant height and root length of susceptible checks despite a nominal decrease observed in highly resistant O. glaberrima accessions (Online Supplementary Table S3 and Fig. 2). The average plant height of PR121 was 57.83 ± 0.13 cm in the nematode infested plot and 79.33 ± 0.27 cm in non-infested conditions showing a reduction of 27.1%. The average plant height of O. glaberrima accessions ranged from 86.0 ± 0.43–134.0 ± 0.50 cm in nematode infested plot and 88.0 ± 0.40–137.0 ± 0.48 under controlled conditions. Root length showed a maximum reduction of 6.4% in O. glaberrima accessions, while PR121 had a reduction of 29.4% in the nematode infested plot. The two accessions (IRGC102196 and IRGC102380) maintained their root length even in RKN infestation.
Figure 2. Effect of M. graminicola on plant height (a) and root length (b) of O. sativa cultivars (Pusa1121, PR116, PR121) and O. glaberrima accessions (IRGC102196, IRGC102538, IRGC102557, IRGC102206, IRGC102356, IRGC102380) in infested and non-infested conditions. Data are means of three replicates for two years. Error bars represent standard deviation. Different letters indicate significant differences between infested and non-infested conditions which were determined by Student's t-test.
Histopathological analysis
The histopathological studies of root galls of susceptible check PR121 showed the presence of a group of enlarged giant cells (thickened cell walls) inside the vascular region of the root cortex (Online Supplementary Figs. S4a and S4b). On the other hand, the giant cells did not develop normally, and little necrosis was observed in the root cortex of resistant O. glaberrrima acc. IRGC102196 (Online Supplementary Fig. S4c). Cross and longitudinal sections of M. graminicola infested roots showed intact vascular bundle (Fig. 3a), and stele (Fig. 3b) in O. glaberrima, whereas necrosis in root cortex and hollow cross-section of mature galls in PR121 was observed (Figs 3c and 3d).
Figure 3. SEM analysis of infested roots from O. glaberrima acc. IRGC102196 shows an intact vascular bundle (a) and stele (b). SEM analysis of infested roots from O. sativa cv. PR121 showing necrotic tissue (c) and hollow mature root gall (d).
Identification of potential SNPs/InDels
Fourteen nematode resistance gene sequences from different crop plants (Online Supplementary Table S1) were aligned with the O. sativa Nipponbare reference genome of rice through the Basic local alignment search tool (BLAST). Only two of the genes viz., Oryzacystatin-I (NC_029256.1) on chromosome 1 and Oryzacystatin-II (NC_029260.1) on chromosome 5 of the O. sativa japonica group showed a significant length of sequence similarity whereas, others had only small regions i.e. <50 bases. Comparison of Oryzacystatin-I and Oryzacystatin–II genes between O. sativa indica group (ASM465v1) and O. glaberrima (GCA_000147395.1) retrieved from Ensembl Plants (https://plants.ensembl.org/species) showed the presence of one SNP and eight SNPs, respectively, between these two species. Further, alignment of genic sequences between PR121 and O. glaberrima acc. IRGC102206 revealed 7 SNPs and 4 InDels (3–6 bp) for the Oryzacystatin-I gene (1610 bp). Only one SNP (G274C) was located in the first exon (127–354 bp), and the remaining 6 SNPs (C126G, A177G, C524T, T601C, C970T, T1057A) were present in the intronic region of Oryzacystatin-I gene. The identified SNPs included six transitions and 18 transversions (Online Supplementary Table S4). A 19 bp insertion (482–500 bp) and 58 bp deletion (989–1046) were detected within the introns of the Oryzacystatin-I gene in both PR121 and IRGC102206. The alignment of the Oryzacystatin-II gene (1517 bp) between PR121 and IRGC102206 showed 19 SNPs and 3 bp InDel (696–698 bp). Out of 19 SNPs, only three SNPs (T163C, G169A, C291T) were located in the first exon (127-387 bp). Similar to Oryzacystatin-I, a 16 bp insertion (525–540 bp) was identified in the intronic region of Oryzacystatin-II compared to the Nipponbare reference gene. Comparative sequence analysis showed more transitions (15) than transversions (7) in the Oryzacystatin-II gene. The identified SNPs/InDels may be associated with the functionality of proteins that might be responsible for conferring resistance in O. glaberrima. It could be explored further to have insights into predicting the role of these genes.
Discussion
The present study has great significance for breeding direct-seeded rice as ground-level water is going down, and today's scenario lays the foundation of agriculture for aerobic rice. In aerobic rice systems, the crop is grown in nonpuddled, non-flooded fields like an upland crop (unsaturated condition) with adequate inputs and supplementary irrigation when rainfall is insufficient (Priyanka et al., Reference Priyanka, Jitesh and Babu2012). Aerobic rice is more vulnerable to RKN due to increased water stress (Das et al., Reference Das, Zhao, De Waele, Tiwari, Shrivastava and Kumar2011). Hence, identifying resistant sources could be valuable for exploiting them in breeding programs of direct-seeded rice. The absence of complete resistance against M. graminicola has been emphasized in earlier studies conducted by different researchers (Plowright et al., Reference Plowright, Coyne, Nash and Jones1999; Soriano et al., Reference Soriano, Espiritu, Schmit, Brar and Reversat1999; Shrestha et al., Reference Shrestha, Uzzo, Wilson and Price2007; Dimkpa et al., Reference Dimkpa, Lahari, Shrestha, Douglas, Gheysen and Price2016; Subudhi et al., Reference Subudhi, Dash, Meher, Mohapatra and Mishra2017). The present study revealed the differential response of O. sativa and O. glaberrima genotypes upon M. graminicola infestation. All O. sativa genotypes were highly susceptible with a reproduction factor >1.5 except for five basmati genotypes which showed a moderately susceptible reaction. However, the accessions of O. glaberrima displayed higher levels of resistance, amongst which three were highly resistant.
The underlying resistance mechanism of O. glaberrima was reported earlier by Petitot et al. (Reference Petitot, Kyndt, Haidar, Dereeper, Collin, Engler, Gheysen and Fernandez2017) through microscopic observation of infested roots and histological analysis of galls in O. glaberrima acc. TOG5681. Penetration and development of M. graminicola juveniles were limited in the resistant acc. TOG5681 as compared to susceptible O. sativa ‘Nipponbare’ rice. The giant cells showed degeneration in the resistant genotype from 15 days of post inoculation (dpi) onwards (Petitot et al., Reference Petitot, Kyndt, Haidar, Dereeper, Collin, Engler, Gheysen and Fernandez2017). The collapse of giant cells and degeneration has also been found in O. glaberrima before J2 developed into adults (Cabasan et al., Reference Cabasan, Kumar, Bellafiore and De-Waele2014). The incompatible interaction between M. graminicola and roots of O. glaberrima might be due to a hypersensitive response that blocks giant cell initiation and its expansion or impair the function of giant cells acting as active transfer cells. Production of phenolic compounds by plants might be associated with the degradation of giant cells and nematode collapsing (Petitot et al., Reference Petitot, Kyndt, Haidar, Dereeper, Collin, Engler, Gheysen and Fernandez2017).
We also observed a higher reduction in shoot and root growth of O. sativa genotypes compared to O. glaberrima accessions. Higher galling in O. sativa might have disrupted the proper supply of micronutrients from roots to shoots, which has compensated to an extent in resistant genotypes (Dangal et al., Reference Dangal, Shrestha, Sharma-Poudyal and Adhikari2009; Pandey et al., Reference Pandey, Nayak and Kar2016). The negative correlation of root gall index and soil nematode population density with plant height and root length of O. sativa genotypes and O. glaberrima accessions was observed. Previous studies also showed a negative and significant correlation between galling with different agronomic traits of plants (Galeng-Lawilao et al., Reference Galeng-Lawilao, Kumar and Waele2018). The results showed that a higher gall index decreased shoot and root growth upon M. graminicola infestation.
The comparative mapping of nematode resistance genes from other crop species showed very low homology with the rice genome, indicating that there might be different resistance genes present in O. glaberrima against M. graminicola. The in silico analysis of Oryzacystatin (Cysteine proteinase inhibitors) genes between O. sativa and O. glaberrima revealed genetic differences for SNPs/InDels that could be putative defensin candidate genes involved in RKN resistance. The expression of serine or cysteine proteinase inhibitors as transgenes has been exploited for resistance to insect pests (Singh et al., Reference Singh, Singh, Kumar, Mittal and Singh2020). However, its use as anti-nematode gene has also been investigated (Ali et al., Reference Ali, Azeem, Li and Bohlmann2017). So far, Oryzacystatin genes have not been explored against M. graminicola. Efforts have been made to understand the genetic variation for these two genes present between O. sativa and O. glaberrima. The SNPs identified between O. sativa and O. glaberrima for these two genes in the present study could be validated for association with nematode resistance in wet-lab experiments. Also, allele mining could be done for these two genes in all the accessions of O. glaberrima to detect favourable haplotypes associated with nematode resistance. It would provide a sight to understand the mechanism of RKN resistance as cross-talk exists between different genes.
The present study holds significance in the identification of three highly resistant donors viz., IRGC102196, IRGC102538 and IRGC102557, for their use against the damaging pathogen of rice M. graminicola. Despite O. glaberrima as a potential resistant source of RKN, its widespread use is limited by its lower yield potential. Therefore, advanced backcross progenies have been developed through interspecific crosses to introgress RKN resistance from African rice species to high-yielding O. sativa cultivars. These backcross progenies are being screened for resistance to the RKN and simultaneously for their use in molecular mapping of genetic loci responsible for M. graminicola resistance to gain sight in crop improvement programs.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S1479262123000965.
Acknowledgements
Funding provided by the Science and Engineering Research Board, Department of Science Technology, India through the project entitled, ‘Genomic assisted mapping and introgression of RKN resistance gene (s) from African rice species O. glaberrima to the Asian rice O. sativa’ (EMR/2016/007539) is gratefully acknowledged.
