White grubs (Coleoptera: Scarabaeidae) associated with Nepalese agriculture and their control with the indigenous entomopathogenic fungus "Metarhizium anisopliae" (Metsch.) Sorokin

White grubs became increasingly difficult pests in Nepal for the last few years. Their infestation has been reported throughout the country and magnitude of the problem has been widespread over the past years. In majority of the farming situation, control of these pests are largely abandoning because of the lack of control over their damages. In general, the management strategy depends primarily on the use of highly poisonous poor graded chemical pesticides. The use of bio-control agents in general and fungal based mycoinsecticides in particular are lacking in the country. The former practices have further aggravated the pest problem resulting in wider reluctances for the cultivation of major cereals as well as cash crops in upland farming. With a view to address these issues, an alternative control measures based on fungal antagonist with the fungus Metarhizium anisopliae in Nepal was sought since 2003 with the initiation and financial support of Helvetas/ Intercooperation through the coordination of Sustainable Soil Management Programme (SSMP-Nepal).
The research was prompted by the serendipitous findings of M. anisopliae and Beauveria bassiana in Nepal from infected white grubs and soils during an exploratory study at Institute of Agriculture and Animal Sciences (IAAS), Rampur. Insect pathogenic fungi, M. anisopliae (green muscardine fungus) and B. bassiana (white muscardine fungus) were identified by a Swiss scientist, Dr Siegfried Keller for the first time in Nepal. With the hope of gearing up of the further works, research plans were drafted, collaborating organizations were identified, and financial supports were solicited and permitted. The research was intensively supported and monitored at various capacities by Dr Siegfried Keller, Senior Scientist, Agroscope FAL Reckenholz, Zurich, Prof. Dr Peter Nagel, University of Basel, Dr Dirk Ahrens, Germany and Dr Philip Kessler, Switzerland through their visits and electronic contacts. This thesis examines the effect of the indigenous insect pathogenic fungus M. anisopliae against white grubs in Nepal through a series of laboratory and field experiments. Each of the stages in the development and optimization of a mass production technique for the field assessment is discussed.
Two kinds of initiatives such as exploratory initiatives and exploitation initiatives were undertaken during the study. Both groups of initiatives are interlinked and mutually contributing to each other. The former initiative includes the search of the indigenous insect pathogenic fungi from the soils and insects mainly from white grub prone areas of Nepal. The activities placed under the exploitation initiatives include development of the technology of production and application of insect pathogenic fungi as a means of white grubs control in particular and pest insects in general. In order to conduct the laboratory work, the Insect Pathology Laboratory with a modest level of facilities has been established within the premises of the Entomology Department of IAAS, Rampur of Tribhuvan University (TU). Several dozens of isolates of the insect pathogenic fungi M. anisopliae and more than half a dozen of B. bassiana were recovered from natural soil and diseased insects using a selective medium and the Galleria bait method (GBM). They have been maintained at IAAS, Rampur and as a security copy at FAL Agroscope Reckenholz, Zurich. Isolation, maintenance, mass production and efficacy tests with virulent strains of M. anisopliae were conducted. Identification of the damaging species of white grubs was one of the pre requisite for the development of effective control measures. Dynamics of the common beetle species were carried out through light traps and field sampling. They were
later identified based on morphological grounds. A three-tired screening strategy: exploration of insect pathogenic fungi in the farmers’ field, pathogenicity assessment under laboratory conditions and its exploitation in farmer’s field in Chitwan, Nawalparasi, Tanahun and Parbat Districts of Nepal has been initiated.
Exploratory study revealed that M. anisopliae is widely distributed in the Nepalese soils and in insects, however, with low density of B. bassiana. Selective medium has proved to be suitable medium for fungus growth and maintenance. Host passages are needed for retaining the virulence. Disease prevalence of M. anisopliae in grub cadaver was between 0 and 2% depending on host origin and species and B. bassiana was found only from a few soil samples. Analysis of soils from different regions showed that M. anisopliae is common and was present in about 50% of the samples irrespective of their origin.
Screening of fungus isolates in time mortality studies indicated that eight isolates gave over 80% infected grubs, sixty five isolates gave over 50-60% infected grubs and rest of the isolates resulted in a low mortality. Five isolates were identified as highly pathogenic (p<0.001) against third instar larvae of Maladera affinis Blanchard in a concentration of 107 spores /ml. Based on infection rates, the fungus isolates M1, M6, M18, M48, and M50 were found aggressive as compared to rest of the strains. The LT50 of all isolates varied between 2-9 weeks, 12 isolates were highly virulent with an LT50 of 2-4 weeks, 34 isolates had a moderate virulence with an LT 50 of 5-6 weeks and 22 isolates had a low virulence. It is interesting to note that, isolates M1 and M6 were found comparatively more virulent because they killed the larvae reasonably shorter period of time as compared to others strains. These five virulent isolates were further studied in dose mortality assays using conidiospores and blastospores in respect to mass production.
The onset of cumulative mortality and mycosis with different dosages suggested that grubs were moderately to highly susceptible to the fungi with all the dosages, however, higher dosages (p< 0.001) were more effective as compared to lower dosages as could be expected. Pathogenicity of conidiospores and blastospores against three different instars of white grubs showed that second instar larvae were more (p<0.001) affected than first and third instars. The information regarding the pathogenicity to different stages of the insect would be helpful in targeting their vulnerable stages. Based on time-mortality and dose-mortality studies, M1 strain of fungus was selected for mass production for field application.
Comparative studies of the fungus production in different types of polybags such as polybags with Swiss origin and Nepali origin as well as studies with solid substrates using peeled kernels of barley, rice and wheat were conducted. In all cases, a total of five virulent strains of M. anisopliae were tested initially and M1 strain was found superior amongst them. Assessment between bag quality convincingly showed the marked difference between the Swiss polybags and Nepali polybags (p<0.001) with a better quality of the fungus in the former types of bags. Similarly, barley kernels are found to be a better substrate for fungus production (p <0.001) than rice and wheat. The fungus colonized grains originating from Nepali bags were heavily contaminated irrespective of the solid substrates. In the same way fungus propagules produced as blastospores were better than conidiospores in terms of purity and grain colonisation. The study has indicated the
opportunity of producing barley grains fully colonized with the fungus as a means of controlling white grubs in Nepal.
The results of the field experiments revealed that the fungus can infect the grubs, however, the infection rate were found very low (15-20%) and this parameter remain insignificant when tested at different dosages. In contrast to the infection rate, the establishment of the fungus after application into the soils was found highly significant (p<0.005) since the colony forming units (CFU) differed greatly while comparing their density before and after application. In general, both the infection rate and fungus density remain shortly until seven weeks and did not lasts at the same extent until crop harvests (thirteen weeks). This result clearly demonstrated that several factors are responsible for the spread and survival of the fungus in the soil. The density of the grub and soil temperature might have attributed the result since the grub density was reduced three months after sampling. In addition to these factors, the virulence of the BCAs, application and assessment methods, and application seasons may play a great role. Based on this information, M. anisopliae can be applied as an important component for white grub management with some modifications. We have to improve our method of production of the fungus, its handling, timing of application and frequencies etc. The persistence and survival of the fungus in the soils are other aspects which need to be considered. Higher mortalities could be achieved using either a white grub’s attractant with any preferable crop root feeding or feeding with grains to lure the white grubs to areas of high spore concentrations, or to position M. anisopliae granules or spores in the soil so that white grubs would pass through them in their normal patterns of movement such as from lower soil layers to upper layers in the spring and vice-versa in summer. The luring materials would have positive effect in attracting the grubs.
Monitoring studies of white grubs clearly indicated that various species of white grubs are involved in crop damages. Normally, the beetles in almost all the studied areas were active during May to July coinciding with (near) crop sowing, early vegetative growth and harvesting stage of maize. In low belt (terai region) of Nepal, the flight of large number of beetle species occurs in two peaks probably because of the short and overlapping generations of the larvae compared to mid hill regions. In mid hill areas, few species of beetles occur regularly in the same crop field, whereas few other beetle species were common at alternate year probably because of the longer life cycle of the larvae. This pattern suggests there might be involvement of univoltine and multivoltine species in the same locality. There are two peaks of occurrence of the annual beetles the first being March-April and second in June-July. In 2004 in Gunganagar research site, the highest catches of the beetle were found in May (5094) followed by April (2324), whereas, very few beetles were caught in Gaindakot research site. At this site, the highest number was 216 and 118 in May and April respectively. Based on their occurrence, Maladera affinis Blanchard being the most frequent (21.2%) followed by Allisonotum simile Arrow (19.2%) in Gaindakot research site. In the same way Adoretus lasiopygus Burmeister was the most frequent species (51.4%) in Gunganagar followed by Anomala dimidiata Hope (6.3%). Similar trends in the occurrence of the beetle were observed in 2005, however, with very low number of catches. Light traps were found one of the effective tools for monitoring phototropic beetles and the information generated from these studies are useful in planning the beetle management programme. Therefore, application of the fungus in any locality should be carried out based on sampling of the white grubs.
In order to understand the natural mortality factors associated in regulating insect population, life table studies and life cycle studies were conducted in 2003/04. These study sites represented low hill area, low mid hill area and mid hill area of Chitwan, Tanahun and Parbat Districts of Nepal respectively. The major objectives of these studies were to know the disturbing factors of white grubs and duration of insect instars and stages so as to plan the microbial control programs in such areas. The life table revealed some natural antagonists such as fungi, M. anisopliae and B. bassiana, and endoparasitic nematode (Mermithidae) coupled with environmental stresses (physical factors) was recorded in suppressing the larval stages. In the same way, different duration of life cycles was observed with different species of beetles involved in different agro-ecological zones. The larval duration of Lepidiota albistigma Burmeister, was found significantly longer (284 days), followed by Maladera affinis Blanchard (58 days) and Xylotrupes gideon L. (57 days) and this parameter was found significantly different (p<0.001) among the species.
Population dynamics in Gunganagar (Chitwan) provided estimates of annual crop losses due to Scarabaeid larvae from 12-35% however, in epidemics this figure raises depending on the season and locality. Farmers may underestimate the role of these soil pests as only 16% of the framers (50 farmers household) surveyed in Gaindakot (Nawalparasi) site mentioned white grubs as a pest, whereas 90% did mention this as major pest in Chitwan. Collection of beetles from digging and light traps since 2003-2005 revealed a large number of beetle species in Nepal. The study so far indicated eighty seven different species of beetles in the study sites. The most important and frequently occurring beetle species were Adoretus lasiopygus Burmeister, Anomala dimidiata Hope, Maladera affinis Blanchard, Heteronychus lioderes Redtenbacher, Anomala bilobata Arrow, Anomala xanthoptera Blanchard, Maladera cardoni Brenske, Idionychus excisa Arrow, Anomala cantori Hope, Mimela silguria Arrow. Based on sampling studies and farmers observations, these species occur lesser or greater extent annually in all the study sites.
The population dynamics through digging indicates that a soil depth up to 20 cm is the most preferable depth for larval activity, whereas eggs are mostly laid up to 15 cm. The pupae and adults are concentrated somehow deeper than the other stages and were mostly found during the winter months. Larval activity in terai conditions was found highest during March-April and June-July. The possible reasons may be due to the availability of the host crops and favorable environment coupled with overlapping generations in the same environment. Similarly, the pupae and adults observed during winter months in the soil and only very few species such as A. dimidiata and few other congregate in tree plants for breeding purposes and majority of them held breeding unnoticing. The knowledge on the overall handling of the fungus, application, virulence, growth and survival of the biocontrol agents in the soil coupled with the pest identity and biology are important aspects for the development of biological control strategies in most of the environments.