Occurrence of Metarhizium spp. isolated from forest samples in South India and their potential in biological control of banana stem weevil Odoiporus longicollis Oliver

Background Thirty-six entomopathogenic fungi (EPF) were isolated from soil and insect cadaver samples, collected from different forest types, viz., wet evergreen, moist deciduous, dry deciduous and scrub type in South India. Partial sequences of two parsimony informative genes ITS and RPB1 were determined under a phylogenetic approach for assessing the genetic diversity. Results Twenty-seven RPB1 gene sequences and 34 sequences of ITS1, 5.8S and ITS2 regions belonging to 36 EPF were analysed for identification and characterization. Four species of Metarhizium viz., M. anisopliae, M. roberstii, M. majus and M. guizhouense were differentiated. The isolates could be grouped into four main clades of 1–5. Most of the fungi appeared to be closely related to M. anisopliae. Based on the colony characters, colour, conidial size and shape, 27 isolates were morphologically identified as M. anisopliae. Seven strains were apparently related to M. robertsii, three isolates were similar to M. majus and the remaining one was identified as M. guizhouense. Morphological studies in congruence with phylogenetic analysis resolved the species diversity. Bioassay studies showed that M. quizhouense, M. majus and M. robertsii were effective against the banana stem weevil Odoiporus longicollis. Conclusions This is the first attempt to study the diversity and occurrence of Metarhizium species in forests of South India. Wet evergreen forest of Aralam in South India was rich in EPF diversity particularly for three species namely, M. quizhouense, M. robertsii and M. anisopliae.

In-vitro Study on the Efficacy of Entomopathogenic Nematodes (Heterorhabditidae and Steinernematidae) for the Control of the Indian Cabbage White, Pieris canidia

Pieris canidia is one of the serious pests of cruciferous crops causing extensive damage to agricultural crops. Entomopathogenic nematodes (EPNs) therefore represent ideal candidates for biological control of the pest. In this study, the efficacy of four indigenous EPNs species, Heterorhabditis indica, Heterorhabditis baujardi, Steinernema sangi, and Steinernema surkhetense from Mizoram was evaluated against P. canidia under laboratory condition. Different concentrations of nematodes, 10, 25, 50, 100, and 200 infective juveniles per larva (IJs/larva) were used to evaluate larval mortality rate, host penetration rate, and progeny production in insect cadaver. All the four EPN species showed high pathogenicity against the pest for different nematode concentrations and observation periods. At a concentration of 200 IJs/larva, cent percent mortality of the pest was recorded at 72 hr post-incubation for S. surkhetense, 96 hr for S. sangi and H. indica, and 120 hr post-incubation for H. baujardi. Based on the values of LC50 and LT50, H. indica was the most pathogenic among the four nematode species while S. sangi was the least pathogenic. After 24 hr of incubation, the LC50 values of H. indica, H. baujardi, S. surkhetense, and S. sangi were 88.60, 98.29, 113.26 and 95.61 IJs/larva, respectively. At 10 IJs/larva, the LT50 values of H. indica, H. baujardi, S. sangi, and S. surkhetense were 88.12, 90.69, 88.102.30, and 88.11 hr, respectively. The study also disclosed that all the four EPN species successfully infect the pest and showed high rate of penetration into the host. Furthermore, all the nematode isolates were capable of producing large numbers of IJs in larval cadaver of P. canidia that significantly varies between the EPN species. The isolate H. baujardi produced the highest number of IJs followed by H. indica, S. surkhetense, and S. sangi. Our findings therefore reveal that all the four EPN isolates have the potential to be developed as biological control agents for P. canidia.

Photorhabdus: a tale of contrasting interactions

Different model systems have, over the years, contributed to our current understanding of the molecular mechanisms underpinning the various types of interaction between bacteria and their animal hosts. The genus Photorhabdus comprises Gram-negative insect pathogenic bacteria that are normally found as symbionts that colonize the gut of the infective juvenile stage of soil-dwelling nematodes from the family Heterorhabditis. The nematodes infect susceptible insects and release the bacteria into the insect haemolymph where the bacteria grow, resulting in the death of the insect. At this stage the nematodes feed on the bacterial biomass and, following several rounds of reproduction, the nematodes develop into infective juveniles that leave the insect cadaver in search of new hosts. Therefore Photorhabdus has three distinct and obligate roles to play during this life-cycle: (1) Photorhabdus must kill the insect host; (2) Photorhabdus must be capable of supporting nematode growth and development; and (3) Photorhabdus must be able to colonize the gut of the next generation of infective juveniles before they leave the insect cadaver. In this review I will discuss how genetic analysis has identified key genes involved in mediating, and regulating, the interaction between Photorhabdus and each of its invertebrate hosts. These studies have resulted in the characterization of several new families of toxins and a novel inter-kingdom signalling molecule and have also uncovered an important role for phase variation in the regulation of these different roles.

Bacterial community profile after the lethal infection of Steinernema–Xenorhabdus pairs into soil-reared Tenebrio molitor larvae

ABSTRACT The host microbiota may have an impact on pathogens. This is often studied in laboratory-reared hosts but rarely in individuals whose microbiota looks like that of wild animals. In this study, we modified the gut microbiota of the insect Tenebrio molitor by rearing larvae in soil sampled from the field. We showed by high throughput sequencing methods that this treatment modifies the gut microbiota so that it is more diversified than that of laboratory-reared insects, and closely resembled the one of soil-dwelling insects. To describe what the entomopathogenic bacterial symbiont Xenorhabdus (Enterobacteriaceae), vectored by the soil-dwelling nematode Steinernema, might experience in natural conditions, we studied the infestation of the soil-reared T. molitor larvae with three Steinernema–Xenorhabdus pairs. We performed the infestation at 18°C, which delays the emergence of new infective juveniles (IJs), the soil-dwelling nematode forms, but which is a temperature compatible with natural infestation. We analyzed by high throughput sequencing methods the composition of the bacterial community within the insect cadavers before the first emergences of IJs. These bacterial communities were generally characterized by one or two non-symbiont taxa. Even for highly lethal Steinernema–Xenorhabdus pairs, the symbiont does not dominate the bacterial community within the insect cadaver.

Dispersal and Repulsion of Entomopathogenic Nematodes to Prenol

Chemosensory cues are crucial for entomopathogenic nematodes (EPNs)—a guild of insect-killing parasitic nematodes that are used as biological control agents against a variety of agricultural pests. Dispersal is an essential element of the EPN life cycle in which newly developed infective juveniles (IJs) emerge and migrate away from a resource-depleted insect cadaver in order to search for new hosts. Emergence and dispersal are complex processes that involve biotic and abiotic factors, however, the elements that result in EPN dispersal behaviors have not been well-studied. Prenol is a simple isoprenoid and a natural alcohol found in association with EPN-infected, resource-depleted insect cadavers, and this odorant has been speculated to play a role in dispersal behavior in EPNs. This hypothesis was tested by evaluating the behavioral responses of five different species of EPNs to prenol both as a distal-chemotactic cue and as a dispersal cue. The results indicate that prenol acted as a repulsive agent for all five species tested, while only two species responded to prenol as a dispersal cue.

Patogenitas cendawan entomopatogen Nomuraea rileyi (Farl.) Sams. terhadap hama Spodoptera exigua Hübner (Lepidoptera: Noctuidae)

The purpose of this research is to study the pathogenicity of Nomuraea rileyi (Farl.) Sams. to eggs and larvae of Spodoptera exigua Hübner (Lepidoptera: Noctuidae).The fungi were isolated from insect cadaver of S. exigua. Egg clusters and several instar larvae with different conidial concentrations (106 – 109 conidia/ml) were used in the experiment. The results of experiment showed that there was no effect of all concentrations of fungi on egg mortality. Mortality of S. exigua larvae was dependent on the instar and conidial concentration. Generally with increasing conidial concentrations, the mortality were also increasing.

THE CONTINUAL FORMING AND CONTRIBUTION OF INFECTIVE JUVENILES PRODUCED VIA ENDOTOKIA MATRICIDA OF ENTOMOPATHOGENIC NEMATODES IN THE FAMILY OF STEINERNEMATIDAE AND HETERORHABDITIDAE

The non-feeding developmentally arrested infective juveniles (IJs) of entomopathogenic nematodes in the family of Steinernematidae and Heterorhabditidae seek out a susceptible insect host and initiate infections. The aim of the research was to examine the continual<br />forming and contribution of IJs produced via endotokia matricida (IJs-EM) of Heterorhabditis bacteriophora, Steinernema glaseri, and S. carpocapsae. The research was conducted at the Laboratory of Nematology of the Saga University, Japan (April 2001-April<br />2002) and the Laboratory of Nematology of the Indonesian Legume and Tuber Crops Research Institute (June 2003-October 2004). The nematode progenies were investigated using the greater wax moth, Galleria mellonella, pre-inoculated with 50 IJs at 25°C.<br />Results showed that three reproductive adult generations were observed at day 18th. There were 135,000, 128,000 and 133,000 IJs per insect cadaver produced in H. bacteriophora, S. glaseri and S. carpocapsae, respectively. Endotokia matricida contributed a higher number of IJs than that of a normal mode of IJs production. The ratios are 81%, 28% and 64% for H. bacteriophora, S. glaseri, and S. carpocapsae of the IJs total production, respectively. Among the generations, the highest contribution of IJs was come from the<br />third adult generation bearing endotokia matricida, i.e., 63%, 24% and 51% for the three nematode species. Although the IJs-EM were more transparent compared to the normal IJs, they were morphologically similar. The results show that endotokia matricida has a pivotal role in a species maintenance and survival strategy of entomopathogenic nematodes in extreme environmental conditions.

THE CONTINUAL FORMING AND CONTRIBUTION OF INFECTIVE JUVENILES PRODUCED VIA ENDOTOKIA MATRICIDA OF ENTOMOPATHOGENIC NEMATODES IN THE FAMILY OF STEINERNEMATIDAE AND HETERORHABDITIDAE

The non-feeding developmentally arrested infective juveniles (IJs) of entomopathogenic nematodes in the family of Steinernematidae and Heterorhabditidae seek out a susceptible insect host and initiate infections. The aim of the research was to examine the continual<br />forming and contribution of IJs produced via endotokia matricida (IJs-EM) of Heterorhabditis bacteriophora, Steinernema glaseri, and S. carpocapsae. The research was conducted at the Laboratory of Nematology of the Saga University, Japan (April 2001-April<br />2002) and the Laboratory of Nematology of the Indonesian Legume and Tuber Crops Research Institute (June 2003-October 2004). The nematode progenies were investigated using the greater wax moth, Galleria mellonella, pre-inoculated with 50 IJs at 25°C.<br />Results showed that three reproductive adult generations were observed at day 18th. There were 135,000, 128,000 and 133,000 IJs per insect cadaver produced in H. bacteriophora, S. glaseri and S. carpocapsae, respectively. Endotokia matricida contributed a higher number of IJs than that of a normal mode of IJs production. The ratios are 81%, 28% and 64% for H. bacteriophora, S. glaseri, and S. carpocapsae of the IJs total production, respectively. Among the generations, the highest contribution of IJs was come from the<br />third adult generation bearing endotokia matricida, i.e., 63%, 24% and 51% for the three nematode species. Although the IJs-EM were more transparent compared to the normal IJs, they were morphologically similar. The results show that endotokia matricida has a pivotal role in a species maintenance and survival strategy of entomopathogenic nematodes in extreme environmental conditions.

Death Becomes Them: Bacterial Community Dynamics and Stilbene Antibiotic Production in Cadavers of Galleria mellonella Killed by Heterorhabditis and Photorhabdus spp.

ABSTRACTInsect larvae killed by entomopathogenic nematodes are thought to contain bacterial communities dominated by a single bacterial genus, that of the nematode's bacterial symbiont. In this study, we used next-generation sequencing to profile bacterial community dynamics in greater wax moth (Galleria mellonella) larvae cadavers killed byHeterorhabditisnematodes and theirPhotorhabdussymbionts. We found that, althoughPhotorhabdusstrains did initially displace anEnterococcus-dominated community present in uninfectedG. mellonellainsect larvae, the cadaver community was not static. Twelve days postinfection,Photorhabdusshared the cadaver withStenotrophomonasspecies. Consistent with this result,Stenotrophomonasstrains isolated from infected cadavers were resistant toPhotorhabdus-mediated toxicity in solid coculture assays. We isolated and characterized aPhotorhabdus-produced antibiotic fromG. mellonellacadavers, produced it synthetically, and demonstrated that both the natural and synthetic compounds decreasedG. mellonella-associatedEnterococcusgrowth, but notStenotrophomonasgrowth,in vitro. Finally, we showed that theStenotrophomonasstrains described here negatively affectedPhotorhabdusgrowthin vitro. Our results add an important dimension to a broader understanding ofHeterorhabditis-Photorhabdusbiology and also demonstrate that interspecific bacterial competition likely characterizes even a theoretically monoxenic environment, such as aHeterorhabditis-Photorhabdus-parasitized insect cadaver.IMPORTANCEUnderstanding, and eventually manipulating, both human and environmental health depends on a complete accounting of the forces that act on and shape microbial communities. One of these underlying forces is hypothesized to be resource competition. A resource that has received little attention in the general microbiological literature, but likely has ecological and evolutionary importance, is dead/decaying multicellular organisms. Metazoan cadavers, including those of insects, are ephemeral and nutrient-rich environments, where resource competition might shape interspecific macrobiotic and microbiotic interactions. This study is the first to use a next-generation sequencing approach to study the community dynamics of bacteria within a model insect cadaver system: insect larvae parasitized by entomopathogenic nematodes and their bacterial symbionts. By integrating bioinformatic, biochemical, and classicin vitromicrobiological approaches, we have provided mechanistic insight into how antibiotic-mediated bacterial interactions may shape community dynamics within insect cadavers.