Status of Meloidogyne graminicola in rice (Oryza sativa L.) and its host range under Bihar conditions

Rice (Oryza sativa) is a staple crop of Bihar, India and various parts of the world. It is a major cereal crop of Bihar State. It contains rich source of carbohydrates (75%) and proteins (7%). -. (the softness of rice due to sarch but not Gluten) Rice protein is also known as Oryzenin. It not only contains carbohydrates and proteins but also lipids, vitamins and minerals. It is the most staple food in the world, with more than one-third of the population playing a competitive role in the food economy and is produced annually in 165 million ha area with 523.9 million tonnes of global annual production . India is the world’s second largest producer of rice, accounting for about 26% of the global production. Other top rice producing countries include Bangladesh, Indonesia and Vietnam (FAO, 2023). This crop is highly vulnerable to several insect pests (Prasad et al., 2006).

Rice root-knot nematode, Meloidogyne graminicola is distributed globally and caused up to 70 % yield loss (Bridge et al., 2005). In India, losses range between 16 and 32 % under irrigated conditions and between 11 and 73 % under flooded conditions (Prasad et al., 1986) and 11% to 73% reported by Soriano et al. (2000). It became a serious issue in the nurseries, upland rice, deep water and irrigated rice and also many parts of India (Prasad et al., 1985; Bridge et al., 2005; Jairajpuri and Baqri, 1991). The Meloidogyne species is highly economic species, causing diseases in many crops (Singh et al., 2019, Singh et al., 2021, Sumita and Vivekananda, 2023) because it is a polyphagous species, but, the species, Meloidogyne graminicola affects only paddy and other graminaceous crops and weeds. It causes typical hook shaped root galls on all the vulnerable hosts. M. graminicola has various host range (Ou, 1972; Sabir and Gaur, 2005) that includes many varieties of paddy and also some common weeds of rice fields. Its occurrence in various host plants of fodder, fruits, ornamentals and weeds, was found by MacGowan and Langdon (1989). It was reported that rice, sorghum, pearl millet, oats and wheat were good hosts of M. graminicola but brinjal, okra, tomato, green gram and barley did not support its multiplication (Dabur et al., 2004). The main hosts of rice root knot nematode in the family,poaceae, includes rice, sorghum, barley, oat and wheat (Vaish et al., 2012).

The most prominent symptoms of M. graminicola on the root system are swollen and hooked root tips which are characteristic for M. graminicola and M. oryzae (Bridge et al., 2005). Typical above ground symptoms include stunting and chlorosis leading to reduced tillers and yield (Narasimhamurthy et al., 2016). The Meloidogyne genus is considered the most important mainly due to its wide host range that includes more than 3,000 species of wild and cultivated plants. M. graminicola is an obligate sedentary endoparasite adapted to flooded conditions. Until recently, M. graminicola was present mainly in irrigated rice fields in Asia, parts of the Americas and South Africa. This pest was included in the EPPO Alert List because of its wide host range and ability to survive during long periods in environments with low oxygen content, represent a threat for rice production in the European Union (Rusinque et al., 2021). Rice root knot nematode (M. graminicola) has emerged as a major threat worldwide and because of its magnitude, has occupied a position of global pest (Narsimhamurthy et al., 2018).

Management of this minuscule crop pest is a herculean task before the nematologists. Traditional practices like crop rotation, fallowing, removing of alternate hosts like weeds have been proven to reduce the nematode population but they are impractical or economically unviable. Three bio-agents (Bacillus pumilus, Bacillus subtilis and Pseudomonas florescence @ 20 g/ m²) have been found effective against this nematode (Kumar et al., 2024). There are very few nematicides available for this nematode management and that too are highly expensive. Although, biological control is a promising alternative, it is still in its infancy. The use of resistant cultivars offers a sustainable solution to manage this shrewd microscopic enemy (De Waele et al., 2013; Yao et al., 2020).

The survey was done during kharif season of 2019 in four districts of Bihar viz., Samastipur, Muzaffarpur, Darbhanga and Vaishali, to find out the infestation of M. graminicola in rice nurseries. The root and soil samples of the crops were brought from the rice field, along with the information of village name, variety, date of sowing, latitude and longitude, crop rotation practices, soil type and seed treatment, gathered from the farmers from 20 fields (Fig 1 and 2). Plants for sampling were selected based on the typical symptoms, i.e. yellowing and stunting of plants. Plants were sampled from 5 spots from each nursery. From each spot, 5 plants were gently uprooted from the depth of 10-15 cm with the help of a scoop. The soil samples, collected from different places in a nursery were kept together and composite representative sample of 200 g of soil along with roots and other plant materials, was collected and packed in the polythene pouches and tagged with  labels.



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Samples were brought to the P.G. laboratory of Department of Nematology, RPCAU, Pusa, Samastipur, Bihar for further processing and data recording. Soil from the roots was removed by gently shaking the plant and the data of root-knot nematode was recorded by the presence of galls with naked eyes. The number of root galls, number of females per root gall, number of eggs and second stage juveniles (J₂) were recorded from the plants. Incidence of root-knot disease was measured by root gall index (1-5 scale), as given by Hartman and Sasser (1985), 1 = no galling; 2 = 1-10 galls; 3 = 11-30 galls; 4 = 31-100 galls; 5 = more than 100 galls/ root system. In the laboratory, nematodes were extracted using Cobb’s sieving and decanting method. The extracted nematodes were killed and fixed in double strength formalin solution and then proceeded for clearing. After clearing in Sienhorst’s solution 1 and 2, permanent slides were prepared with glycerol for further observation.

Frequency of distribution was calculated using below mentioned formula:

 

 
Nematode culture
 
The pure culture of M. graminicola population, used in this study, was maintained in the field laboratory of Nematology, RPCAU, Pusa, from single egg mass of infected rice roots. The infected plants were uprooted and egg masses were carefully detached from the galled roots. The egg masses were stirred with 0.5 % NaOCl solution and kept in Modified Baermann Funnel Technique (MBFT) for hatching and approximately 1000 J2s were used to infect paddy (susceptible variety, PB 1121) roots in pots. After 30 days of infection, nematode females and egg masses were extracted (Hussey and Barker, 1973). Using perineal patterns (Eisenback et al., 1980), identity of M. graminicola was confirmed. Egg masses were collected (Hussey and Barker, 1973) from the infected roots at 30 days post-inoculation (dpi), surface sterilized with 0.1% HgCl₂ for 1 min, followed by rinsing in sterile water. Egg masses were kept for hatching in a double-layered tissue paper placed on a wire mesh in a Petri plate containing sterile distilled water. Freshly hatched J₂s were used for the inoculation.
 
Screening of rice root knot nematode against various host ranges
 
The experiment was conducted in pots to study the host differential test of Meloidogyne graminicola on different varieties of rice (viz., Rajendra Sarsawati, Rajshree, BPT 5204, Rajendra Neelum, Rajendra Prabhat, Rajendra Sahbhagi, Rajendra Abhishek, Rajendra Shweta and Rajendra Vaidehi), coarse cereal, bajra (HHB-67) and millets like, barnyard millet (Dholi-1), finger millet (BR-407, RAU-3, RAU-8) and Foxtail millet (RAU-1) with vegetables like brinjal (BR-112), onion (Kharif) and tomato (Selection-120), sown in infested soil, collected from Pusa farm of rice field. Each treatment  was replicated three times and the experiment was conducted in CRD design. The plants were uprooted 30 days after inoculation, each seedling was packed in polythene pouch and tagged with replication and treatment number and brought to the laboratory. Roots were properly and gently washed in running tap water and then staining was done using 0.1% fuchsin acid lactophenol. The stained roots were observed under binocular microscope for the following observations: number of root-knots, number of females, number of juveniles, number of eggs and juveniles per egg mass and root-knot index.

During survey of the rice nurseries, the highest nematode population was observed in Samastipur (5000 nematodes/ 200 cc of soil) and lowest nematode population was found in Dharbhanga (2200 nematodes/200 cc of soil). Highest number of root knots/plant was observed in Vaishali district (55) and lowest was found in Muzaffarpur (20 root knots /plant). Highest root knot index was (3.66) in Vaishali district and lowest root knot index was 2.33, found in Darbhanga district. Highest number of eggs and juveniles was found in Samastipur district (62.55 per egg mass) and lowest was found in Muzaffarpur district (20.0 per egg mass). Highest number of females was observed in Samastipur (3.33) and lowest was found in Dharbhanga (0.66) district, as shown in Table 1. The results obtained in the present study are similar to Khan et al., (2019) who conducted survey in major rice growing districts of Uttar Pradesh and concluded that highest frequency of disease in rice fields was observed in Aligarh (44.6%), followed by Muzaffarnagar, Shahjahanpur and Kheri Lakhimpur (29.3, 28.0 and 27.4 respectively). Similarly Narsimhamurthy et al., (2017) recorded population density of M. graminicola in different districts of Karnataka in the range of 586.33 to 841.00 J2/100 cc of soil. Rana et al., (2016) evaluated 50 germplasm of basmati rice, out of which Pusa 1637-18-7-6-20 was found to be resistant with scale 2, while, two germplasm, Shaan (Hybrid) and UPR 3805-12-2-7 were evaluated to be susceptible with scale 4. Jabbar et al., (2015) observed its prevalence from last three years in rice-wheat cropping system in Faisalabad and Chiniot districts of Punjab in Pakistan and results showed 20% infestation rate in Faisalabad and 17% in Chiniot district.



Table 2, reveals that, among rice varieties, significantly highest root knots per plant and root knot index was recorded in BPT-5204 (13.0 and 3.0) whereas lowest number of root knots and root knot index was noticed in Rajendra Saraswati (7.66 and 2.0). The varieties, Rajshree and Rajendra Prabhat have shown the reaction at par. Similarly, when observation on number of females per root knot, number of juveniles per root knot and number of eggs and juveniles per egg mass were noticed, same trend was observed i.e., maximum number in BPT-5204 and minimum in Rajendra Saraswati.



Among millets, Finger millet (BR-407), Barnyard millet (Dholi-1) and Bajra (HHB-67) are at par showing appearance of root knots (6.67, 6.00 and 6.67 respectively), where the nematode parasite produced maximum females being 4.33 on Barnyard millet (Dholi-1) whereas Finger millet (RAU-3), (RAU-8) and Foxtail millet (RAU-1) did not reveal any gall on the roots, exactly same trend was shown when we observe number of females per root knot, number of juveniles per root knot and number of eggs and juveniles per egg mass among millets which were tested.

Except onion, no other vegetables revealed any sign of host parasite interaction on the host i.e., neither knots were found on the roots, nor the females have developed inside the root. Obviously, eggs were not found and no juveniles were reported. In onion crop, number of root knots per plant, number of juveniles per root knot and number of eggs and juveniles per egg mass were up to the tune of 7.67, 3.33, 4.00 and 4.33, respectively. This result is in conformity with Dabur et al., (2004) who reported that rice, sorghum, pearl millet, oats and wheat were good hosts of M. graminicola but brinjal, okra, tomato, green gram and barley did not support its multiplication. Similar finding was obtained by Dhurvey et al., (2019) as maximum number of galls per plant (36.66 and 57) in paddy varieties, Pusa basmati and JR 503 respectively while the 6 cultivars, IR64, JR75, Poornima, Shyamla, PS3 and Madhuri showed resistant reactions against M. graminicola and the result is also supported by Subudhi et al., (2017) who found that the paddy varieties viz., Satabdi, Sabita, PR114, Savitri, Geetanjali, Tapaswini, Pooja, Sambhamahasuri, Ratna etc. were found to be highly susceptible having score 5. Khanish, Uday, Basmati-370, Jogen, Dinesh, Vanaprava and ASD-16, were found to be moderately resistant against this nematode.

From the result of current research, it is inferred that the nematode parasite multiplied best in the rice variety, BPT-5204, followed by Rajendra Prabhat, Rajshree, Rajendra Vaidehi, Rajendra Abhishek, Rajendra Neelum, Rajendra Sahbhagi and Rajendra Saraswati (Fig 3 to Fig 12 ). Among millets, the nematode multiplied only on finger millet (BR-407), Barnyard millet (RAU-1) and Bajra (HHB-67), whereas among vegetables, only onion supported nematode multiplication but to a lower extent. Salalia (2015) when studied 14 populations of M. graminicola from India, observed that six varieties of rice, two kharif weeds and brinjal were found as hosts, tomato (hybrid PKM 1) was non-host for all the populations, but status of pearl millet and sorghum was not clearly discernible.



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From the present study it is concluded that, infestation of M. graminicola in nursery of paddy, was maximum in Samastipur district and minimum in Darbhanga district. The host status of M. graminicola on different varieties of paddy, different millets and in vegetables showed that all varieties of rice were infested in different intensity and cause losses. Among the millets, foxtail millet (RAU-1), finger millet (RAU-3 and RAU-8) did not show any nematode infestation, but, barnyard millet (Dholi-1), finger millet (BR-407) and bajra (HHB-67) did get the infestation. And for the vegetables, only onion showed the nematode infestation and rest two vegetables, brinjal and tomato did not reveal gall symptoms. So, it can be concluded that the paddy varieties are good host of M. graminicola while some millets also showed the positive response. Vegetables still do not prove a good host of M. graminicola. This nematode has a very high adaptation of new hosts as reported by many workers, specially different paddy verieties and many weeds. So, the management options should be explored with antagonistic plants or through trap crops, which are the best management options. This nematode is highly adaptive to the host and so, it is expanding its host range, covering many crops. So, the future research may be done on some other graminaceous crops and weeds to get the knowledge of host diversity to get the management insights regarding crop rotation and antagonistic crops.

The authors gratefully acknowledge Dr. Rajendra Prasad Central Agricultural University, Samastipur, Pusa, Bihar, India for providing the laboratory and other important facilities and ICAR for granting National Talent Scholarship (NTS) for the present research.

There is no any conflict of interest between the authors.