Cloning and characterization of resistance gene analogs from under-exploited plant species

Genomic DNA sequences sharing homology with NBS region of resistance gene analogs were isolated and characterized from Pongamia glabra, Adenanthera pavonina, Clitoria ternatea and Solanum trilobatum using PCR based approach with primers designed from conserved regions of NBS domain. The presence of consensus motifs viz., kinase 1a, kinase 2, kinase 3a and hydrophobic domain provided evidence that the cloned sequences may belong to the NBS-LRR gene family. Conservation of tryptophan as the last residue of kinase-2 motif further confirms their position in non-TIR NBS-LRR family of resistance genes. The Resistance Gene Analogs (RGAs) cloned from P. glabra, A. pavonina, C. ternatea and S. trilobatum clustered together with well- characterized non-TIR-NBS-LRR genes leaving the TIR-NBS-LRR genes as a separate cluster in the average distance tree constructed based on BLOSUM62. All the four RGAs had high level of identity with NBS-LRR family of RGAs deposited in the GenBank. The extent of identity between the sequences at NBS region varied from 29% (P. glabra and S. trilobatum) to 78% (A. pavonina and C. ternatea), which indicates the diversity among the RGAs

Relative tolerance and expression of resistance to phosphine in life stages of the rusty grain beetle, Cryptolestes ferrugineus

Cryptolestes ferrugineus is a serious cosmopolitan pest of stored products. Frequent and indiscriminate usage of phosphine has caused the development of high levels of resistance to this fumigant. As there are few alternatives, it is imperative that resistance to phosphine is managed. Effective management requires knowledge of key factors driving the rate of selection. One of the most important factors is the response of each resistance genotype to phosphine, especially heterozygotes. Moreover, it is important to understand the expression of resistance in all life stages as all stages are subjected to selection during fumigation. We determined the relative tolerance and resistance levels to phosphine in all life stages of homozygous parental strains (susceptible and resistant) and their F1 progeny (heterozygous) and estimated relative dominance of resistance within life stages over 48 h. In susceptible insects, relative tolerance was highest in eggs followed by pupae, then adults which had about the same tolerance as larvae. In homozygous resistant insects, the order of tolerance was adult = egg > pupae > larvae and in heterozygotes larvae > eggs > pupae > adults. All life stages expressed resistance with resistance ratios highest in adults > pupae > larvae > eggs. At LC50, resistance was incompletely recessive in eggs, pupae and adults and incompletely dominant in larvae. Eggs and adults were also incompletely recessive at LC95, but larvae were completely dominant and pupae were incompletely dominant. Our data showed that a proportion of heterozygotes in all life stages, the major carriers of resistance in the field, will survive at very high concentrations, particularly in the egg stage, forming a nucleus for reinfestation or dispersal of resistance

In vitro Assessment of Neonicotinoids and Pyrethroids against Tea Mosquito Bug, Helopeltis antonii Sign. (Hemiptera: Miridae) on Guava

The tea mosquito bug (TMB), Helopeltis antonii, is an emerging pest of horticultural crops, specially on guava and moringa. Insecticides are indispensable component for the management of insect pests. Exploration of new molecules with shortest waiting period may pave way for managing TMB in fruit and vegetable crops with nil/low residue. Until now there are no recommended insecticides available under Central Insecticides Board & Registration Committee (CIB&RC) against TMB on guava. In view of the above facts, new molecules with a low waiting period and are recommended by CIB&RC on tea, viz., Clothianidin 50% WDG, Thiacloprid 21.7% SC, Bifenthrin 10% EC, and Thiamethoxam 12.60% + Lambda-Cyhalothrin 9.5% ZC, were chosen and evaluated against TMB under in vitro condition. Clothianidin 50% WDG recorded the highest mortality of 100.00 per cent at 72 hours after treatment (HAT), and the lowest LC50 value (0.328 ppm, fiducial limits: 0.144-0.515 ppm) and LT50 value (10.49 h, fiducial limits: 5.444-14.551 h), followed by Thiamethoxam 12.60% + Lambda-Cyhalothrin 9.5% ZC, Thiacloprid 21.7% SC, and Bifenthrin 10% EC. The results showed that the Clothianidin 50% WDG and Thiamethoxam 12.60% + Lambda-Cyhalothrin 9.5% ZC, were highly effective, with the lowest LC50 and LT50 values. Since TMB occurs from new flushing to fruiting stage of guava, a minimum of two sprays are mandatory to have quality fruit yield. Hence, application of Clothianidin 50% WDG followed by Thiamethoxam 12.60% + Lambda-Cyhalothrin 9.5% ZC on need basis will help to reduce the impact of TMB on guava

Pathogenicity, Ovicidal Action, and Median Lethal Concentrations (LC50) of Entomopathogenic Fungi against Exotic Spiralling Whitefly, Aleurodicus dispersus Russell

Biological control using entomopathogenic fungi could be a promising alternative to chemical control. Entomopathogenic fungi, Beauveria bassiana (Balsamo) Vuillemin, Metarhizium anisopliae (Metschnikoff) Sorokin, Lecanicillium lecanii (Zimmerm.) Zare and Gams, and Paecilomyces fumosoroseus (Wize) Brown and Smith, were tested for their pathogenicity, ovicidal effect, and median lethal concentrations (LC50) against exotic spiralling whitefly, Aleurodicus dispersus Russell. The applications were made at the rate of 2 × 109 conidia mL−1 for evaluating the pathogenicity and ovicidal effect of entomopathogenic fungi against A. dispersus. The results of pathogenicity test showed that P. fumosoroseus (P1 strain) was highly pathogenic to A. dispersus recording 100% mortality at 15 days after treatment (DAT). M. anisopliae (M2 strain) had more ovicidal effect causing 37.3% egg mortality at 8 DAT. However, L. lecanii (L1 strain) caused minimum egg hatchability (23.2%) at 10 DAT as compared to control (92.6%). The lowest LC50 produced by P. fumosoroseus (P1 strain) as 8.189 × 107 conidia mL−1 indicated higher virulence against A. dispersus. Hence, there is potential for use of entomopathogenic fungi in the field conditions as an alternate control method in combating the insect pests and other arthropod pests since they are considered natural mortality agents and are environmentally safe

Development of microsatellite markers and a preliminary assessment of population structuring in the rice weevil, Sitophilus oryzae (L.)

The rice weevil, Sitophilus oryzae is one of the primary pests of stored grains worldwide. To develop and implement an effective integrated pest management strategy, an understanding of the population structuring of this destructive pest is vital. In this study we used Illumina paired-end sequencing to develop S. oryzae species-specific microsatellite markers, and used these markers to conduct a preliminary assessment of population structuring in four populations of S. oryzae from three countries (Australia, China, and USA). 7,635,996 raw sequencing reads were produced, with 11,794 microsatellites detected and 214,257 primer options designed. 48 microsatellite markers were selected for further validation, with 10 markers amplifying consistently across the four S. oryzae populations. These markers displayed a high level of polymorphism overall (6.67 alleles/locus), though this was slightly lower within populations (3.10-4.88 alleles/locus). We used the markers to conduct a preliminary assessment of genetic structuring among the four S. oryzae populations: three laboratory cultures (New South Wales, Queensland, and Santai) and a field collected population from Kansas. Analyses suggest high levels of genetic differentiation between the sample locations, with a global F of 0.239, and pairwise F values ranging from 0.100 to 0.395. Bayesian clustering analyses suggest these four populations formed four distinct clusters, with a similar pattern identified by Principal Coordinate Analysis. These microsatellite markers, together with our preliminary population genetic analyses, will provide a valuable resource for population genetic research, and contribute to effective integrated pest management strategies in the future

Phenotypic and molecular analyses in rice weevil, Sitophilus oryzae (Linneaus) (Coleoptera: Curculionidae): identification of a super kdr mutation, T929I, conferring resistance to deltamethrin

BACKGROUND The rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae) is a cosmopolitan pest of stored cereal grains and other commodities globally. Infestations caused by S. oryzae makes grains unsuitable for consumption, processing, and export. Deltamethrin, a synthetic pyrethroid insecticide, is widely used in major grain storages in India as a prophylactic treatment to control this pest. However, recurrent use of this insecticide had led to genetic resistance in S. oryzae, questioning its ongoing use at the current recommended concentration. RESULTS Dose response analysis of resistant (Delta-R) and susceptible (Lab-S) strains of S. oryzae collected from grain storages across southern India, revealed that Delta-R was 134-fold more resistant than the Lab-S at median lethal concentration (LC50). A concentration of 180 ppm over 48 h effectively discriminated 16 resistant field populations from Lab-S with per cent resistance ranging from 8.72% to 75.86%. Exposing all the resistant populations to 1000 ppm over 48 h identified 12 populations with strongly resistant individuals and confirmed the existence of two distinct resistance phenotypes, ‘weak’ and ‘strong’ in S. oryzae. Furthermore, sequence analysis of the voltage-gated sodium channel (vgsc) gene in Delta-R identified a single target site mutation, T929I conferring resistance in S. oryzae. CAPS (Cleaved Amplified Polymorphic Sequence) marker analysis of this allele confirmed that frequency of resistance is high (up to 0.96) supporting the results of phenotypic analysis. CONCLUSION Both phenotype and molecular marker analyses clearly demonstrated that deltamethrin at 180 and 1000 ppm can be used to discriminate weakly and strongly resistant populations in S. oryzae, respectively. Resistance diagnostics based on the mutation, T929I, supports our phenotypic data and indicates that resistance to deltamethrin in S. oryzae is prevalent in southern parts of India, stressing the need to identify a synergist or suitable alternatives

Molecular characterization of the invasive fall armyworm, Spodoptera frugiperda (J.E. Smith) feeding on finger millet, Eleusine coracana (L.) Gaertn

Genetically, the maize fall armyworm (FAW, Spodoptera frugiperda J.E. Smith) consists of two strains viz., corn (C) strain mainly feeding on maize, sorghum, cotton, pulses, etc., and rice (R) strain feeding on rice crop and other grasses. The present study was carried out to identify the strains of FAW collected from finger millet crop at three different locations viz., Morappur (Dharmapuri district), Vridhachalam (Cuddalore district) and Salem (Salem district) of Tamil Nadu, India. PCR–RFLP profile of mitochondrial cytochrome oxidase I fragment exhibited the presence of both ‘C’ and ‘R’ strains of FAW feeding on finger millet. Out of ten samples, 6, 8 and 8 samples assumed ‘R’ strain identity, whereas, 4, 2 and 2 samples assumed ‘C’ strain identity in the locations of Morappur, Vridhachalam and Salem, respectively. Sequence analyses of mtCOI region of FAW feeding on finger millet showed nucleotide variations in eight positions. The molecular identity of S. frugiperda was ranged from 98 to 100% with previously deposited sequences in the NCBI GenBank database

Genetic diversity and its geographic structure in Sitophilus oryzae (Coleoptera; Curculionidae) across India – implications for managing phosphine resistance

The rice weevil, Sitophilus oryzae, is a serious global pest of stored grains. Fumigation with phosphine gas is the primary control method for S. oryzae, but the indiscriminate and prolonged use of phosphine gas has led to the development of heritable resistance. Developing and implementing an effective phosphine resistance management strategy for S. oryzae relies on an understanding of its genetic diversity and any structuring of that diversity geographically. We therefore sequenced the mitochondrial cytochrome c oxidase subunit I gene from 143 S. oryzae specimens collected from 37 locations across India, and from that assessed the genetic diversity of the species and its phylogeographic structuring. In addition, we compared the genetic diversity in Indian S. oryzae populations (the hypothesised origin of this beetle) to global populations. Genetic diversity was low in Indian S. oryzae, with only eight haplotypes (including two very common haplotypes) identified. The low level of mitochondrial diversity observed in this species appears typical of stored product pests, perhaps suggesting that low mitochondrial diversity is associated with repeated phosphine fumigations, which may eliminate low frequency haplotypes. The genetic diversity of S. oryzae in India is, however, higher than in many other countries, though comparable levels were identified in China. There was no evidence of population genetic structure across India, with most haplotypes found in three of the broad biogeographic regions. This lack of phylogeographic structuring indicates significant gene-flow across India, most likely through the incidental anthropogenic transport of this relatively poor (or reluctant) flyer. The major practical implication is that phosphine resistance management for S. oryzae needs to be dealt with country wide, as populations are not isolated