Characterization and phylogenetic analysis of an entomopathogenic Bacillus cereus strain WGPSB-2 (MTCC 7182) isolated from white grub, Anomala dimidiata (Coleoptera: Scarabaeidae)

White grubs (Coleoptera: Scarabaeidae) are cosmopolitan and polyphagous insect pests of agricultural crops, forests and pastures around the world. The lack of an environmentally sound approach for white grub management has prompted the exploration and detection of a novel microbial biocontrol agent against these sub-terranean insect pests. In this study we describe the isolation, establishment of pathogenesis, biochemical characterization and phylogenetic analysis of an entomopathogenic Bacillus cereus strain WGPSB-2 (MTCC 7182), isolated from an atrophied pupa of Anomala dimidiata, collected from the N.W. Indian Himalayas. The sequencing and subsequent comparison of the 16S rDNA revealed that the strain has100% similarity with Bacillus cereus sequences. Phylogenetic analysis of the 16S rDNA sequence revealed that the isolate is closely related to Bacillus thuringiensis and Bacillus sphaericus. In vitro bioassays showed that the isolate was able to infect and cause 92 and 67% mortality in second instar larvae of Anomala dimidiata and Holotrichia seticollis, respectively. The infected larvae exhibited bacterial septicemia like symptoms and mortality occurred between the third and ninth weeks after inoculation. The culture has been granted the accession number MTCC 7182 by the Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Chandigarh, India.     

Virulence of entomopathogenic nematodes to larvae of the guava weevil, Conotrachelus psidii (Coleoptera: Curculionidae), in laboratory and greenhouse experiments

The guava weevil, Conotrachelus psidii, is a major pest of guava in Brazil and causes severe reduction in fruit quality. This weevil is difficult to control with insecticides because adults emerge over a long period, and larvae develop to the fourth-instar inside the fruit and move to the soil for pupation. We assessed the virulence of entomopathogenic nematodes to fourth-instar larvae in soil by comparing their susceptibility to nine species or strains: Heterorhabditis bacteriophora HP88, H. baujardi LPP7, and LPP1, H. indica Hom1, Steinernema carpocapsae All and Mexican, S. feltiae SN, S. glaseri NC, and S. riobrave 355. In petri dish assays with sterile sand at a concentration of 100 infective juveniles (IJs) of a given nematode species/strain, larval mortality ranged from 33.5 to 84.5%, with the heterorhabditids being the most virulent. In sand column assays with H. baujardi LPP7, H. indica Hom1, or S. riobrave 355 at concentrations of 100, 200, and 500 IJs, mortality was greater than the control only for H. baujardi (62.7%) and H. indica (68.3%) at the highest concentration. For H. baujardi LPP7 in a petri dish assay, the time required to kill 50 and 90% of the larvae (LT₅₀ and LT₉₀) for 100 IJs was 6.3 and 9.9 days, whereas the lethal concentration required to kill 50 and 90% of the larvae (LC₅₀ and LC₉₀) over 7 days was 52 and 122.2 IJs. In a greenhouse study with guava trees in 20-L pots, 10 weevil larvae per pot, and concentrations of 500, 1000 or 2000 IJs, H. baujardi LPP7 caused 30 and 58% mortality at the two highest concentrations. These results show that H. baujardi is virulent to fourth-instar larvae and has potential as a biological control agent in IPM programs.     

Laboratory evaluation of entomopathogenic fungi against the white grubs,Holotrichia oblita and Anomala corpulenta (Coleoptera: Scarabaeidae) from the field of peanut,Arachis hypogaea

We evaluated the pathogenicity of nine isolates of entomopathogenic fungi, including six isolates of Metarhizium anisopliae, one of Beauveria bassiana, and two of Beauveria brongniartii, against eggs and various larval instars of two scarab-beetle species, Holotrichia oblita and Anomala corpulenta, under laboratory conditions. The fungal isolates differed in pathogenicity. Generally, the isolates were more pathogenic to A. corpulenta than to H. oblita. Some of the isolates prevented egg hatching and caused larval death. Isolates M2-2 and Br5-8 caused 39 and 27% egg mortality, respectively, and produced 23 and 24% viewable fungal-infection rates in H. oblita. Three isolates had no significant effect on egg hatchability. Three isolates (Br5-8, Br232818, and M200614) caused about 40% mortality in H. oblita first instars. In A. corpulenta, all isolates except M200614 caused more than 60% egg mortality, and M2-2, Br232818 and Br5-8 caused egg-infection rates greater than 50%. M2-2 caused 47% infection and 100% mortality in first-instar larvae of A. corpulenta, while Br5-8 and Br232818 yielded over 80% mortality of the larvae. The three most virulent isolates, M2-2, Br232818 and Br5-8, were selected for further bioassays against second- and third-instar larvae. In addition, seven graduated concentrations of a Br5-8 conidial suspension were assayed against H. oblita second instars. Larval mortality was positively correlated with fungal dosage, and the LC₅₀ was 4.49×10⁶ conidia/mL. These three virulent isolates may be used to prevent H. oblita and A. corpulenta larval infestations in field crops.     

Susceptibility of Hoplia philanthus (Coleoptera: Scarabaeidae) larvae and pupae to entomopathogenic nematodes (Rhabditida: Steinernematidae, Heterorhabditidae)

Biological control potential of nine entomopathogenic nematodes, Heterorhabditis bacteriophora CLO51 strain (HbCLO51), H. megidis VBM30 strain (HmVBM30), H. indica, Steinernema scarabaei, S. feltiae, S. arenarium, S. carpocapsae Belgian strain (ScBE), S. glaseri Belgian strain (SgBE) and S. glaseri NC strain (SgNC), was tested against second-, and third-instar larvae and pupae of Hoplia philanthus in laboratory and greenhouse experiments. The susceptibility of the developmental stages of H. philanthus differed greatly among tested nematode species/strains. In the laboratory experiments, SgBE, SgNC, HbCLO51 and HmVBM30 were highly virulent to third-instar larvae and pupae while SgBE was only virulent to second-instar larvae. Pupae were highly susceptible to HbCLO51, HmVBM30, SgBE and SgNC (57–100%) followed by H. indica and S. scarabaei (57–76%). In pot experiments, HbCLO51, SgBE and S. scarabaei were highly virulent to the third-instar larvae compared to the second-instar larvae. Our observations, combined with those of previous studies on other nematode and white grub species, show that nematode virulence against white grub developmental stages varies with white grub and nematode species.     

Effectiveness of entomopathogenic nematodes Steinernema feltiae and Heterorhabditis bacteriophora on the Colorado potato beetle Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) under laboratory and greenhouse conditions

In laboratory and greenhouse studies, the invading ability, virulence, and mortality caused by Stinernema feltiae and Heterorhabditis bacteriophora were compared. After one and two days of exposure to either nematode species, the mortality of Colordo potato beetle (CPB) Leptinotarsa decemlineata larvae at different instars, third and fourth, was recorded and the number of nematodes invading cadavers was more than the number of nematodes inside the larvae at the late last instar (one day before pre-pupa). Two concentrations, 250 and 500 IJs/dish, infective juvenile nematodes/0.5 ml were tested on different CPB larval instar. S. feltiae was more effective, with fourth instar rather than third and late last instar. On the other hand, H. bacteriophora showed a very weak effect with L. decemlineata. Also it was clear that S. feltiae was more effective and faster than H. bacteriophora: more than 70% of larvae were killed within 24 hours compared with H. bacteriophora which killed 40% of larvae within 48–72 hours. A significant difference in invading efficiency was observed with concentration 2500 IJs/pot in the greenhouse test. The number of adult females found in the cadavers of L. decemlineata larvae was always higher than the number of males. Foliage application of S. feltiae and H. bacteriophora resulted in a significant reduction of the number of damaged leaves and a lower index of damage compared with that in the control. We conclude that S. feltiae has significant potential and can help in the management of the Colorado potato beetle.     

嗜菌异小杆线虫侵染后暗黑鳃金龟和大黑鳃金龟幼虫脂肪体和中肠组织超微结构观察

【目的】探究昆虫病原线虫嗜菌异小杆线虫沧州品系 Heterorhabditis bacteriophora strain Cangzhou侵染对蛴螬脂肪体和中肠的影响,进一步明确其对蛴螬的致病机理。【方法】采用透射电镜技术,观察暗黑鳃金龟 Hololtrichia oblita (Faldermann)和大黑鳃金龟 H. parallela Motschulsky 2龄幼虫被嗜菌异小杆线虫沧州品系侵染后其脂肪体和中肠组织的病理变化。【结果】血腔注射感染期病原线虫嗜菌异小杆线虫沧州品系悬浮液24和48 h后,观察发现暗黑鳃金龟和大黑鳃金龟2龄幼虫脂肪体和中肠的组织结构均按时序逐渐发生变化,起初表现为脂肪球变形或变小,颜色变浅,脂肪体细胞和中肠细胞内质网、线粒体肿胀,中肠微绒毛变形脱落等现象,48 h后包裹脂肪球的膜结构破裂,脂肪体细胞和中肠细胞线粒体破裂,内质网数量减少,中肠微绒毛大量脱落,同时核内染色质大量解离,核膜破裂。【结论】经昆虫病原线虫嗜菌异小杆线虫沧州品系处理后,暗黑鳃金龟和大黑鳃金龟两种金龟甲2龄幼虫脂肪体和中肠细胞均出现明显的病理变化过程,这是嗜菌异小杆线虫高效致死蛴螬的原因之一。本研究可为昆虫病原线虫作为一种生物防治手段在蛴螬的综合防治中更好地发挥作用提供理论依据。     

Comparison of aerial conidia and blastospores from two entomopathogenic fungi against Diaphorina citri (Hemiptera: Liviidae) under laboratory and greenhouse conditions

This study compared the insecticidal activity of liquid culture-produced blastospores and solid substrate-produced aerial conidia of Beauveria bassiana GHA and Isaria fumosorosea ARSEF3581 strains against Diaphorina citri adults. Insects exposed to 10⁷ propagules/ml in a spray residue contact leaf bioassay died within 6 days at 25°C, with no significant differences between fungal treatments. At higher concentrations (10⁸ propagules/ml), I. fumosorosea conidia killed psyllids faster compared to its blastospore formulation, i.e. 4 versus 5 days, respectively. In greenhouse tests, the same treatments applied to infested citrus plants (2?×?10⁶ spores/ml) all significantly reduced the number of nymphs compared with the untreated controls over 3 weeks; however, only I. fumosorosea blastospores significantly reduced the number of F1 adult psyllids when compared with controls. Similar results were observed in the follow-up greenhouse test, where I. fumosorosea blastospores were the most effective treatment overall, reducing D. citri populations by about 60% after 21 days; by contrast, imidacloprid killed almost 100% of psyllids within a week in both tests. Fewer psyllids exhibited mycosis in the greenhouse (i.e. ≈20 versus?≥?87% in the laboratory). This is the first report comparing both conidial and blastospore formulations of B. bassiana and I. fumosorosea for the control of a psyllid pest. Field testing is required to determine how successful different spore formulations might be under various environmental conditions.