Characterization of rice rhizospheric Bacillus thuringiensis as biocide against leaf folder (Cnaphalocrocis medinalis), stripe stem borer (Chilo suppresalis), plant pathogens, and plant growth promotion traits.

The incidence of rice pests like the rice yellow stem borer (Scirpophaga incertulas Walker) and the rice leaf folder (Cnaphalocrocis medinalis Guenee) can be influenced by a variety of factors, including planting dates, planting methods, and weather parameters. The present investigation was undertaken to study the effect of planting dates and methods associated with weather parameters on the incidence of the rice yellow stem borer and leaf folder. The experiment was conducted at the Rice research farm of RPCAU, Pusa, in Randomized Block Design during the years 2019 and 2020 with rice variety ‘Rajendra Mahsuri 1.’ The planting dates were categorized as early planting (15th June), timely planting (5th July) and late planting (25th July) at 20-day intervals. The influence of weather parameters was correlated with the stem borer and leaf folder damage, and they were analysed using the SPSS Package. The results revealed that the early transplanted rice crops had the lower pest incidence recording, 12.39 per cent, dead hearts/white ear due to yellow stem borer and 7.49 percent leaf damage due to incidence of rice leaf folder as compared to late transplanted rice crop which recorded 21.75 per cent DH/WE (Dead Heart/White Ear) due to incidence of yellow stem borer and 13.91 percent DL (Damaged Leaves) due to rice leaf folder. Mean percentage of tiller damage was significantly higher in puddled direct seeding (14.67%) as compared to direct seeding (11.77%) followed by timely transplanting (4.40%) whereas the incidence of leaf folder in different planting methods mean percentage leave damage was significantly higher in timely transplanting (15.39%) as compared to direct seeding (12.83%) followed by puddled direct seeding (11.72%). Maximum and minimum temperature, wind speed and morning relative humidity had a negative effect on stem borer per cent dead heart and white ear head and leaf folder concerned, maximum temperature, morning relative humidity, evening relative humidity and rainfall had positive correlation at different planting dates whereas, maximum and minimum temperature, evening relative humidity had a positive effect on stem borer per cent dead heart and white ear head and leaf folder concerned, maximum temperature and morning relative humidity and evening relative humidity had positive correlation at different planting methods.. The findings of the study revealed that the planting dates and methods greatly influenced the magnitude of pest incidence in rice crops. The study further revealed that early and normal transplanting can reduce the insect pest incidence in the rice crop, particularly rice stem borer and rice leaf folder and farmers can harvest a good yield. It is obvious that delayed transplanting and other methods of transplanting would have more severe pest incidence and, thereby, the huge grain yield losses. Among the parameters, relative humidity and rainfall played a major role, which explained the maximum variability on yellow stem borer and leaf folder incidence at different methods and planting dates.

Herbivore-induced defense responses are often specific, whereas plants could induce distinct defense responses corresponding to infestation by different herbivorous insects. Brown plant hopper (BPH) Nilaparvata lugens, a phloem-feeding insect, and rice leaf folder (LF) Cnaphalocrocis medinalis, a chewing insect, are both specialist herbivores on rice. To characterize the distinct resistance primed by prior damage to these two specialist herbivores, we challenged rice plants with two herbivores during vegetative growth of parent plants and assessed plant resistance in subsequent ratoons. Here, we show that LF and BPH induce different suites of defense responses in parent rice plants, LF induced higher level of JA accumulation and OsAOS, OsCOI1 transcripts, while BPH induced higher accumulation of SA and OsPAL1 transcripts. Moreover, an apparent loss of LF resistance was observed in OsAOS, OsCOI1 RNAi lines. Ratoon plants generated from parents receiving prior LF infestation exhibited higher jasmonic acid (JA) levels and elevated levels of transcripts of defense-related genes associated with JA signaling, while ratoon generated from parents receiving prior BPH infestation exhibited higher salicylic acid (SA) levels and elevated levels of transcripts of defense-related genes associated with SA signaling. Moreover, previous LF infestation obviously elevated ratoons resistance to LF, while previous infestation by BPH led to enhanced resistance in ratoons to BPH. Pre-priming of ratoons defense to LF was significantly reduced in OsAOS and OsCOI1 RNAi plant, but silencing OsAOS and OsCOI1 did not attenuate ratoons resistance to BPH. These results suggest that infestation of two specialist herbivores with different feeding styles in parent crop led to distinct defense responses in subsequent rations, and the acquired resistance to LF in ratoons is associated with priming of jasmonic acid-dependent defense responses.

Remote sensing of rice leaf folder damage using ground-based hyperspectral radiometry

Simple SummaryThe transcriptomic and metabolomic differences in rice leaves after infestation by the rice leaf folder Cnaphalocrocis medinalis were investigated for better understanding of the mechanisms of rice defenses against this species. The results suggest that C. medinalis infestation can induce rapid and precise defense responses involved in many primary and secondary metabolic processes in rice leaves, and the jasmonic acid (JA)-dependent signaling pathway plays vital roles in the response of rice plants to this pest species. These results provide comprehensive insights into the defense system of rice to the rice leaf folder and may facilitate the development of insect-resistant rice varieties by identifying molecular targets for selection.Interactions between plants and insect herbivores are important determinants of plant productivity in cultivated and natural agricultural fields. The rice leaf folder (Cnaphalocrocis medinalis) causes tremendous damage to rice production in Asian countries. However, little information is available about how rice plants defend themselves against this destructive pest at molecular and biochemical levels. Here, we observed the transcriptomic and metabolomic differences in rice leaves after 0, 1, 6, 12, and 24 h of being fed by C. medinalis using RNA sequencing and metabolome profiling. Transcriptional analyses showed that gene expression responds rapidly to leaf folder infestation, with the most significant transcriptional changes occurring within 6 h after the initiation of feeding. Metabolite abundance changed more slowly than gene expression. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that the rice transcriptional response to infestation involved genes encoding protein kinases, transcription factors, biosynthesis of secondary metabolites, photosynthesis, and phytohormone signaling. Moreover, the jasmonic acid-dependent signaling pathway triggered by leaf folder herbivory played a vital role in rice defense against this pest. Taken together, our results provide comprehensive insights into the defense system of rice to this species and may inform the development of insect-resistant rice varieties.

Rice plant is attacked by many insect-pests of which yellow stem borer (dead heart at vegetative stage and white ear head at reproductive stage) and leaf folder are considered as prime devastator, responsible for major economic loss. To combat this problem, resistant varieties of rice are required for better production as well as better productivity. Host plant resistance is very much effective in integrated pest management (IPM) system, where negligible pesticidal hazard is present as well as environmental safety, low cost farming by without or minimum pesticide application and proper identification of resistant varieties for selection as parent in crossing programme to develop resistant varieties in future. The 78 rice varieties were screened out against Scirpophaga incertulas (yellow stem borer) and Cnaphalocrocis medinalis (rice leaf folder) of rice during Kharif, 2010-2012 at Rice Research Station, Chinsurah, Hooghly, under Government of West Bengal. The experiment was carried out to determine the level of resistance in early, mid-early, medium and late duration rice varieties, and shallow and semi-deep rice varieties. The pest infestation level was determined by counting dead heart, white ear head and damaged/folded leaves. The experimental results revealed that the early duration variety viz. Narendra 97 and IR 50, the mid-early duration variety i.e. IR 64 and IET 17904 proved resistant against both yellow stem borer (dead heart) and leaf folder. The medium duration variety, Ranjit was highly resistant against both yellow stem borer (dead heart) and leaf folder, and the variety, Pratiksha showed a fair degree of resistance against both yellow stem borer and leaf folder; while Jarava, the late duration variety exhibited high degree resistance against yellow stem borer (dead heart and white ear head). Besides, the varieties, Sashi and Giri recorded very low level of infestation of both lepidopteran pests while shallow and semi-deep variety such as Sashi was also considered resistant against the noxious pests.

The rice leaf roller or leaf folder, Cnaphalocrocis medinalis (Guenee) (Lepidoptera: Pyralidae), is a serious pest of rice crop in Pakistan. The present study was carried out to evaluate the virulence of the entomopathogenic fungi (EPF) Beauveria bassiana, Verticillium lecanii, and Metarhizium anisopliae against the third instar of C. medinalis larvae. Larvae were exposed to fungi under controlled conditions at the available commercial concentration (1 × 108 conidia ml−1). The results showed 73.33, 57.78, and 74.44% mortality rates in the in vitro assay and 56.67, 41.11 and 52.78% in the greenhouse assay of B. bassiana, V. lecanii, and M. anisopliae, respectively. The maximum mycosis from cadavers of C. medinalis was observed at B. bassiana treatment in the in vitro assay (70%) and in the greenhouse assay (53.78%). The maximum sporulation from C. medinalis cadavers was observed at B. bassiana treatment in the in vitro assay (144.67 conidia ml−1) and in greenhouse assay (96.67 conidia ml−1). These results favor the alternative use of EPF in organic rice production for management of C. medinalis.

Natural parasitism of rice yellow stemborer, Scirpophaga incertulas (Walker) and leaffolder, Cnaphalocrocis medinalis (Guenee) in organically as well as conventionally grown basmati rice (Pusa Basmati 1121) was recorded at farmers’ field during 2012. Different stages of stemborer and leaffolder were collected and observed under laboratory conditions for the emergence of parasitoids to record natural parasitism. The parasitism in the eggs, larvae and pupae of rice stemborer and leaffolder ranged from 1.67 to 30.91 and 1.86 to 28.87 per cent in organic and conventional basmati rice, respectively. Egg parasitoids, Trichogramma chilonis, T. japonicum, Telenomus sp. were recorded from the eggs of rice stemborer. Braconid wasps, Stenobracon nicevillei and Bracon sp were recorded from the larvae of stemborer. Tetrastichus sp and Brachymeria sp were the pupal parasitoids with per cent parasitism ranging from 5.09 to 5.36 and 2.83 to 3.17 in conventional and organic fields. Larval parasitoids, Bracon sp and Cotesia sp were recorded from rice leaffolder and the parasitism ranged from 5.88 to 6.02. Pupal parasitoids, Tetrastichus sp and Brachymeria sp were recorded with parasitism ranging from 4.08 to 5.59 per cent.

Entomopathogenic fungal biocides are preferred for environment friendly sustainable management of insect pests due to their host specificity and harmlessness to non-target insects. Plant growth promotion (PGP) functions of the entomofungi are also important attributes but hitherto insignificantly explored. Therefore, virulence of 17 natural fungal entomocides (Cordyceps, Beauveria, Metarhizium, Nomuraea, Fusarium, Verticillium, Trichoderma and Paecilomyces spp.) were evaluated for pathogenicity against five rice pests (brown plant hopper (Nilaparvata lugens) and green leaf hopper (Nephotettix virescens) nymphs, leaf folder (Cnaphalocrosis medinalis) and yellow stem borer (Scirpophaga incertulas) larvae and swarming caterpillar (Spodoptera mauritia), respectively), and PGP traits of the potent leaf folder pathogens. Among the fungi, only the leaf folder pathogens (3 isolates each of Beauveria and Metarhizium spp.) infected > 50% (80-90%) larvae but other fungi were ineffective as infected < 50% (0-47%) insects. Besides, the leaf folder pathogens exhibited diverse PGP traits such as organic/inorganic phosphate solubilization (104.7-236.4µg/ml), and siderophore, ammonia, hydrogen cyanide (HCN), indole production etc. Restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), simple sequences repeat (SSR) and internal transcribed spacers (ITS) analysis ascertained strain identity and genetic (inter and intra-specific) diversity among the potent biocides Beauveria and Metarhizium spp. The virulent natural fungal pathogens of rice pests with polyvalent PGP traits may be prospected for rice growth promotion and biocontrol of leaf folder.

The rice leaf folder,Cnaphalocrocis medinalisGuenée (Lepidoptera: Crambidae), an important foliage feeding insect in rice,Oryza sativaL. (Poaceae), is widely distributed throughout Asia, causing frequent outbreaks and considerable yield losses. Mutants of the rice strain Samba Mahsuri (MSM) represent unique plant material and are explored here for the first time for resistance to the rice leaf folder. Stringent rapid field screening for two consecutive seasons led to the identification of 10 lines as moderately resistant, with low damage area as well as low damage score. Upon assessment of various morphological traits potentially contributing to the resistance, damage area was negatively correlated with leaf length and positively with leaf width. Among the biochemical traits, leaf nitrogen content was positively correlated with leaf folder damage whereas silica was negatively correlated. Oviposition preference by leaf folder adult moths and larval settling preference were low on MSM 139 and MSM 127. We also observed that leaf folder larvae took more time for leaf selection (3.1 min) and making a primary fold (41.5 min), with more head swings (846) in moderately resistant MSM lines. Similarly, in these lines, leaf folders showed a low intrinsic rate of increase (rm = 0.15 females per female per day), low weekly multiplication rate (2.85), and low net reproductive rate (R0 = 137.5 females per female per generation) with increased generation time (T = 32.8 days) and doubling time (4.5 days), indicating their relatively poor population growth potential. Overall, the moderately resistant mutant lines we identified showed reduced ovipositional preference and larval settling preference, extended larval developmental period with low survival, reduced primary fold length, and an increased number of binds and head swings with a low intrinsic rate of increase resulting in reduced population build‐up. These lines possessing a good genetic makeup have a great potential to be used in breeding programs for the development of leaf folder resistant varieties.

The jasmonic acid (JA) pathway plays a key role in plant defense responses against herbivorous insects. CORONATINE INSENSITIVE1 (COI1) is an F-box protein essential for all jasmonate responses. However, the precise defense function of COI1 in monocotyledonous plants, especially in rice (Oryza sativa L.) is largely unknown. We silenced OsCOI1 in rice plants via RNA interference (RNAi) to determine the role of OsCOI1 in rice defense against rice leaf folder (LF) Cnaphalocrocis medinalis, a chewing insect, and brown planthopper (BPH) Nilaparvata lugens, a phloem-feeding insect. In wild-type rice plants (WT), the transcripts of OsCOI1 were strongly and continuously up-regulated by LF infestation and methyl jasmonate (MeJA) treatment, but not by BPH infestation. The abundance of trypsin protease inhibitor (TrypPI), and the enzymatic activities of polyphenol oxidase (PPO) and peroxidase (POD) were enhanced in response to both LF and BPH infestation, but the activity of lipoxygenase (LOX) was only induced by LF. The RNAi lines with repressed expression of OsCOI1 showed reduced resistance against LF, but no change against BPH. Silencing OsCOI1 did not alter LF-induced LOX activity and JA content, but it led to a reduction in the TrypPI content, POD and PPO activity by 62.3%, 48.5% and 27.2%, respectively. In addition, MeJA-induced TrypPI and POD activity were reduced by 57.2% and 48.2% in OsCOI1 RNAi plants. These results suggest that OsCOI1 is an indispensable signaling component, controlling JA-regulated defense against chewing insect (LF) in rice plants, and COI1 is also required for induction of TrypPI, POD and PPO in rice defense response to LF infestation.

Jasmonate-induced accumulation of anti-herbivore compounds mediates rice resistance to the leaf folder Cnaphalocrocis medinalis. The rice leaf folder (LF), Cnaphalocrocis medinalis, is one of the most destructive insect pests in the paddy field. LF larvae induces leaf folding and scrapes the upper epidermis and mesophyll tissues reducing photosynthesis and yield in rice. Identifying plant defense pathways and genes involved in LF resistance is essential to understand better this plant-insect interaction and develop new control strategies for this pest. Jasmonate (JA) signaling controls a plethora of plant defenses against herbivores. Using RNA-seq time series analysis, we characterized changes in the transcriptome of wild-type (WT) leaves in response to LF damage and measured the dynamics of accumulation of JA phytohormone pools in time-course experiments. Genes related to JA signaling and responses, known to mediate resistance responses to herbivores, were induced by LF and were accompanied by an increment in the levels of JA pools in damaged leaves. The accumulation of defense compounds such as phenolamides and trypsin proteinase inhibitor (TPI) also increased after LF infestation in WT but not in JA mutant plants impaired in JA biosynthesis (aoc-2) and signaling (myc2-5). Consistent with all these responses, we found that LF larvae performed better in the JA mutant backgrounds than in the WT plants. Our results show that JA signaling regulates LF-induced accumulation of TPI and phenolamides and that these compounds are likely an essential part of the defense arsenal of rice plants against this insect pest.

A field trial was conducted to evaluate Lambda-cyhalothrin new formulation emulsion-in-water (EW), which indicated that Lambda-cyhalothrin applied at 15 g a.i. ha −1 was more effective against rice leaf folder (Cnaphalocrocis medinalis) as compared to the standard checks. In terms of cumulative reduction of pest incidence, Lambda-cyhalothrin treatments were found to be superior to the standard checks. Lambda-cyhalothrin 11.4% EW was identified as a superior formulation with respect to environmental hazards. Water is being used as solvent to formulate it compared to primitive practice of using naphthalene-based low volatile organic compound products for formulating emulsifiable concentrates. Hence, the dose of 15 g a.i/ha can be recommended for the effective management of rice folder with added advantages of environmental aspects. K e y words: Lambda-cyhalothrin, rice leaf folder, EW, EC, Cnaphalocrocis medinalis

Endospheric microbiome of unexplored wild rice, Oryza brachyantha (OB), was studied. Endogenous heterotrophic, spore forming, spore-crystal forming, nitrifying, denitrifying, P solubilizing bacteria, actinomycetes, and fungi in leaves, stems and roots were (0.07–4.48, 0.06–4.08, 5.00–4.00, 0.56–1.40, 0.06–0.40, 0.07, 0.03 and 0.14–8.00) x10 2 cfu/g, respectively and endo-colonizers were 5–6 times more in roots than other plant parts. Phenotypic characters, cry+/cyt+ in 6 SCF and 16S rDNA phylogeny of 2 potent SCF ascertained their identity as Bacillus thuringiensis (Bt). For the first time, 6 polyvalent Bt with biocidal and plant growth promotion (PGP) functions were identified from the wild rice OB. All endo-Bt possessed diverse anti-lepidopteran cry genes which predicted their virulence against rice leaf folder (LF), Cnaphalocrocis medinalis Guenee. Only cry2 or cyt primers amplified expected sized amplicons from Bt 18, 20 and 21. Bt18 and Bt21 killed 60% LF larvae in vitro by 2.65x104and 7.56x104 cfu/mlandtheformer onewas morevirulent. Theyproducedsiderophore, amylase, protease etc. which would induce natural tolerance of OB against bacterial and fungal pathogens. Besides, the Bt had other PGP traits too viz., nitrate reduction, ammonia production, P solubilization etc. Therefore, the endo-microbiome of OB would modulate endospheric functionalities, promote growth and development, and impart natural endurance to OB against pests and diseases.

Silicon (Si) amendment to plants can confer enhanced resistance to herbivores. In the present study, the physiological and cytological mechanisms underlying the enhanced resistance of plants with Si addition were investigated for one of the most destructive rice pests in Asian countries, the rice leaf folder, Cnaphalocrocis medinalis (Guenée). Activities of defense-related enzymes, superoxide dismutase, peroxidase, catalase, phenylalanine ammonia-lyase, and polyphenol oxidase, and concentrations of malondialdehyde and soluble protein in leaves were measured in rice plants with or without leaf folder infestation and with or without Si amendment at 0.32 g Si/kg soil. Silicon amendment significantly reduced leaf folder larval survival. Silicon addition alone did not change activities of defense-related enzymes and malondialdehyde concentration in rice leaves. With leaf folder infestation, activities of the defense-related enzymes increased and malondialdehyde concentration decreased in plants amended with Si. Soluble protein content increased with Si addition when the plants were not infested, but was reduced more in the infested plants with Si amendment than in those without Si addition. Regardless of leaf folder infestation, Si amendment significantly increased leaf Si content through increases in the number and width of silica cells. Our results show that Si addition enhances rice resistance to the leaf folder through priming the feeding stress defense system, reduction in soluble protein content and cell silicification of rice leaves.

Hyperspectral detection of rice damaged by rice leaf folder (Cnaphalocrocis medinalis)