Novel Sources of Combined Resistance Against Rice Root-Knot Nematode and Brown Spot Disease in Oryza rufipogon

Rice root-knot nematode (RRKN,Meloidogyne graminicola)and brown spot (BS,Drechslera oryzae) are serious constraints on the quality of rice grains, particularly under direct-seeded rice conditions in many parts of the world. Developing rice varieties resistant to RRKN and BS will be the most effective and environmentally friendly management strategy. A total of 93 and 58Oryzarufipogonaccessions were screened against RRKN and BS, respectively, for two years under artificial inoculation conditions. Among the 93O. rufipogonaccessions,only 1 accession showed a highly resistant (HR) reaction, while 12 were found to be resistant (R). Similarly, among the 58O.rufipogonaccessions, 19 were found to be R to BS. Furthermore,based on a two-year evaluation, eight selectedO. rufipogonaccessions were screened against combined inoculation of RRKN and BS, leading to the identification of two accessions showing R to both pathogens, while five accessions exhibited a moderately resistant (MR) to both RRKN and BS, and one accession showed MR to RRKN and R to BS. These accessions can be utilized in breeding program to develop rice varieties resistant to RRKN and BS and map the loci/genes governing these traits.

The practice of cultivating rice under direct-seeded conditions is emerging as an important method due to the water-intensive nature of rice farming, leading to the over-exploitation of water resources (Kaur and Singh, 2017). However, RRKN and BS are limiting factors in the widespread adoption of this method (Kaur and Singh, 2017). RRKN functions as a metabolic sink in hypertrophied root tissues, obtaining photosynthates for growth and development from other sections of the plant via the root system (McClure, 1977). Yellowing, dwarfing/stunting, and the production of galls on the roots of rice plants are the symptoms induced by RRKN infection. The yield losses due to RRKN varied from 16% to 32% in India during different years(Ravindra et al, 2015). A survey conducted in Punjab, India,during 2016-2017 revealed that RRKN is present in 42% of rice nursery beds and in 22.2% of rice fields (Singh, 2017). BS,caused by the fungal pathogenDrechslera oryzae, is known worldwide and has occurred in epiphytotic conditions in different parts of India. This pathogen caused massive losses in Bengal during 1942-1943 (Imran et al, 2020). BS of rice is also referred as the ‘poor man’s disease’ as it typically occurs in areas with water and nitrogen-deficient soils (Barnwal et al,2013). In India, this disease causes heavy yield losses, up to 6%at low to moderate severity levels, and 90% to complete yield loss at moderate to high severity levels, depending on the presence and full functioning of all components of the disease triangle (Imran et al, 2020). The pathogen attacks the rice crop from the seedling to milky stage, and the symptoms include dark brown or purplish brown dots on the coleoptile, leaf sheath, leaf blade, and glume, with the leaf blades and glumes being the most affected. These spots later converge or combine, and the affected leaf dries, giving a burnt appearance of infected rice field. Along with dark spots on the leaves, this fungus also causes browning of rice grains (seed discoloration), significantly impacting the vigour of rice seedlings (Zulkifi et al, 1991).

However, resistance to RRKN is not commonly available in cultivated rice germplasms and most of the Asian cultivated rice genotypes are found to be susceptible (S) to this pathogen(Berliner et al, 2014; Devi, 2014; Dimkpa et al, 2016; Subudhi et al, 2017). Wild relatives of rice are reservoirs of immense genetic variation that have been used to improve resistance to diseases and insect pests in cultivated rice (Sun et al, 2001; Brar and Singh, 2011). Among the wild relatives of rice,O. rufipogonis an ancestral species of cultivated rice. This species is the donor of many important traits, including resistance to several biotic and abiotic stresses (Brar and Singh, 2011; Bhatia et al,2017). In the past, many studies have been performed to identify resistance against RRKN and BS in wild species andO.glaberrima(Soriano et al, 1999; Brar and Singh, 2011), and found that some accessions ofO. glaberrimaand a wild riceO.longistaminataare known to be the donors of RRKN resistance.In addition, a fewO. rufipogonaccessions have been identified as resistant to RRKN (Weerapat and Chongkid, 1990; Hada et al,2020). Tolerance to BS has been identified in four accessions ofO. nivara(Goel and Bala, 2006). However, more intensive efforts are required to identify novel sources of resistance tothese two pathogens. Punjab Agricultural University (PAU),India, maintains a collection of over 1000 accessions of wild rice, includingO. rufipogon, which can become important candidates to uncover higher-level and durable resistance against RRKN and BS.

Table 1. Disease reaction of Oryza rufipogon accessions under artificial inoculation conditions and percent reduction in growth parameters.

Here, we screened a set of 93O.rufipogonaccessions againstM. graminicolain 2018 and 2019 (Table S1). A high-yielding cultivar PR126, recommended by PAU for direct-seeding but highly susceptible (HS) to RRKN, was included as the S check.We observed significant variability among the 93O. rufipogonaccessions in response to RRKN infection, with considerable variation in all RRKN-related traits (Table S2). Of the 93 accessions, no galls were observed in IR93070 (Fig. S1-A),while 12 accessions showed 1%-25% galls, 17 accessions had 26%-50% galls, 44 accessions had 51%-75% galls and 19 accessions had 76%-100% galls on roots (Table 1). Root galling index (RGI) was calculated based on the percentage of galls observed on the roots of each accession. The RGI inO.rufipogonaccessions ranged from 1 to 5, while it was 5 in PR126 (Fig. S1-B). Similarly based on the RGI, IR93070 showed HR to RRKN, while 12 accessions were R and 17 showed MR reaction (Table 1).

The RRKN population was higher in S accessions compared with R accessions (Table S1). The highest RRKN population in the soil was observed inO. rufipogonaccession IR83810 and PR126 (3.06 nematodes/g soil), while the lowest was in accession IR93070 (0.23 nematodes/g soil), which exhibited an HR reaction. Besides, the RRKN population showed a high reproduction factor (Rf ＞ 2) in HS accessions and PR126. The S accessions had Rf values between 1 and 2, while the R accessions had Rf ＜ 1 (Table S1).

Further, based on the observations in infected and non-infected soil, reductions in shoot length, shoot weight, and root length were observed inO. rufipogonaccessions due to RRKN infection (Fig. S2). The maximum decrease in shoot length (＞ 25.65%) was observed inO. rufipogonaccession CR100381. Similarly, the maximum reduction in shoot weight(51.07%) was observed in CR100015, and the maximum reduction (46.38%) in root weight was found in IR100597. In contrast, no significant differences in shoot length, shoot weight,and root length were observed in RRKN-resistant accessions,grown in infected and non-infected soils. There was only a 0.08% reduction in shoot length, a 2.87% reduction in shoot weight, and a 2.72% reduction in root length in infected plants compared with non-infected IR93070. HS accessions reactions to RRKN exhibited yellowing and stunting symptoms, while R accessions remained healthier.

The wild rice relatives are enriched with novel genetic resources, which can be utilized for improving disease resistance in cultivated rice (Brar and Singh, 2011). This study identified 12O. rufipogonaccessions showing R and 1 showing HR reactions toRRKN, indicating the presence of resistant sources in this species. TheseO. rufipogonaccessions are the new potential sources in rice breeding programs. The development of fertile crosses and the opportunity to transfer the resistance through two- or three-backcrosses are the additional advantages for identifying resistance to RRKN inO. rufipogon, compared withO. glaberrimaandO. longistaminata, the known donors.The RRKN-resistant accessions showed negligible reductions in plant growth such as shoot length, shoot weight, and root length based on comparison in infected and non-infected soils.However, significant reductions in these parameters were observed in S and HSO. rufipogonaccessions inM. graminicolainfected soil as reported in earlier studies by Pandey et al (2016).This may be attributed to the reason that HS genotypes allow the juveniles to enter their roots, attain maturity, and reproduce further, while resistant plants suppress their development, and thus do not allow the reproduction (Karssen and Moens, 2006).

Similarly, a set of 58O. rufipogonaccessions (Table S1) was screened for resistance to BS in the years 2018 and 2019. The traits considered for the evaluation of BS resistance showed significant variations among theO. rufipogonaccessions (Table S2). TheO. rufipogonaccessions exhibited differential reactions to BS, as indicated by disease incidence ranging from 0 to 6.33.The lowest or negligible BS incidence was observed in accessions IR105400 and IR105420 (Fig. S1-C), while the highest BS incidence (6.33) was observed in accession IR81996.PR126 showed the highest disease incidence (8.00) (Fig. S1-D).On the basis of disease incidence, the accessions were categorized into four groups: R, MR, MS, and S (Table 1). Of the 58O. rufipogonaccessions, 19 were found to be R, 21 as MR,and 18 as MS to BS (Table 1).

On the basis of observations in infected and non-infected conditions, shoot length and shoot weight were found to be affected by BS infection (Fig. S3). Shoot length and shoot weight were observed to be significantly higher in R accessions compared with S accessions. There were negligible differences in shoot length and shoot weight in R accessions in infected and non-infected plants. However, accessions exhibited a 2%-9%reduction in shoot length, while MS accessions experienced a 4%-12% reduction. The maximum reduction in shoot weight(39.84%) was observed in accession CR100035A with an MS reaction, while the minimum reduction (0.84%) was observed in the accession IR104727 with an R reaction. The maximum reductions in shoot length (44.55%) and shoot weight (56.57%)were observed in PR124 (Fig. S3).

Various researchers have screened rice cultivars for BS in the past and have found variable levels of resistance. Pannu et al(2006) found two cultivated varieties (PR111 and Jaya) with the lowest disease severity of 4.3% and 4.2%, respectively. Magar(2015) evaluated 14 varieties and found disease severity ranging from 21.73% to 58.07%. Arshad et al (2008) observed only 1 resistant genotype among 17 genotypes. Similarly, out of 25 varieties, 4 varieties, namely NDR-359, CR-1, CR-2, and N-18,were found to be R to BS (Alam et al, 2016). During a survey conducted by Pak et al (2017), BS of rice was the predominant fungus found in 13 out of a total of 15 sampling sites and across all 5 locations surveyed in Australia. The infection of BS was also detected in a few wild rice accessions, includingO.australiensis,in the survey,indicating the wide prevalence of this disease. All these studies point to put more efforts to identify diverse sources that could exhibit higher resistant levels to BS.In our study, we have identified 18O. rufipogonaccessions displaying a higher resistant level, indicating that this species harbors favorable genetic variations for resistance to BS.

In addition, reductions in shoot length and shoot weight were also observed in MS and S accessions ofO. rufipogonin BS-inoculated plants, while negligible reduction was observed in R accessions. Characteristic symptoms of BS in the field included oval and eye-shaped spots with a conspicuous dark brown dot in the center and a light brown margin. These spots on the screened rice cultivars consisted of dead tissues and were most commonly observed on the leaves. Kranz et al (1978)reported that the presence of these spots could reduce the leaf area of the plant, which ultimately affects the rice’s photosynthetic activities. The large spots appearing on these varieties may influence the respiratory activity of the plant by bringing changes in the physical and chemical equilibrium of the protoplasm (Kranz et al, 1978). This could result in reduced growth and yield of the affected plants.

Wild species or distant relatives of crop plants may harbor useful variations for multiple important traits. For example,wheat-rye translocations are known to confer resistance to multiple traits, such as resistance to powdery mildew and rusts(Hysing et al, 2007; Zhou et al, 2012). Jansky and Rouse (2003)discovered an interspecific hybrid potato clone that was resistant to five different diseases. Fetch et al (2003) evaluated accessions of the wild progenitor of barley (Hordeum spontaneum) for resistance to six fungal pathogens and observed that resistance to the majority of them was present at high frequencies. TheMi-1gene introduced from wild tomato (Solanum peruvianum) to cultivated tomato (S. lycopersicum) confers resistance against three species of root knot nematodes i.e.M. arenaria,M.incognita, andM. javanica(Smith, 1944; Dropkin, 1969). Later,this gene was also found to be resistant to potato aphid(Macrosiphum euphorbiae), tomato psyllid (Bactericerca cockerelli) and whitefly (Bemisia tabaci) (Rossi et al, 1998;Nombela et al, 2003; Casteel et al, 2006). We have identifiedO.rufipogonaccessions that showed resistance to RRKN and BS.These accessions are novel sources of resistance against both pathogens and should be used in breeding programs. We are further developing a mapping population from one of these accessions to map and transfer resistance genes into the cultivated rice background.

ACKNOWLEDGEMENTS

The authors are thankful to the Head of the Department of Plant Pathology, Punjab Agricultural University, India, for providing the required research facilities and in-house funding for carrying out this study. We also thank Dr Kumari NEELAM, School of Agricultural Biotechnology, Punjab Agricultural University,India, for providing seeds ofO. rufipogon.

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science;http://www.ricescience.org.

File S1. Methods.

Fig. S1. Resistant reactions to rice root-knot nematode and brown spot in accessions.

Fig. S2. Interaction plots ofOryza rufipogonaccessions and PR126 upon infection to rice root-knot nematode.

Fig. S3. Comparative shoot length and shoot weight of infected and non-infected plants with brown spot pathogen.

Table S1. Rice root-knot nematode and brown spot traitsofOryza rufipogonaccessions and controls.

Table S2. Statistical analysis for rice root-knot nematode and brown spot inOryza rufipogonaccessions under infected conditions.