ENTOMOPATHOGENS AS NATURAL ENEMIES AND BIOLOGICAL CONTROL AGENTS © I W. Mudita PS IHPT Fakultas Pertanian Undana Citation: Mudita, I W. 2006. Pengendalian Hayati: Pengendalian Hama, Patogen, dan Gulma dengan Musuh Alami. Bahan Ajar PS IHPT Fakultas Pertanian Universitas Nusa Cendana, Kupang

DEFINITION Entomopathogens are microbes that parasite insects by which the host insects become paralyzed and finally died The parasitized insects are called hosts The Entomopathogens can infect insect naturally, in this case they are natural enemies When Entomopathogens are intentionally cultured then released to control a particular insect pests, they are biological control agents

MAJOR GROUP OF ENTOMOPATHOGENS Bacteria: Bacteria are microscopic prokaryotes, without well-defined nucleus or organelles, have a structurally distinct cell wall. Fungi: Fungi are eukaryotes, with a well-defined nucleus and organelles, characterized by criticized cells. The cells are formed into filaments or hyphae together forming a mycelium. Viruses: Viruses are sub-microscopic, intracellular, obligate pathogens. They can neither move nor metabolise. They consist of a template nucleic acid with or without a protein coat, which is also called an inclusion or occlusion body. Protozoa: These are unicellular organisms classified in several phyla. Nematodes: Nematodes are unsegmented, worm-like organisms with a tough outer cuticle. Nematodes are larger than other entomopathogenic organisms and can usually be seen with the naked eye. They can act as vectors for entomopathogenic bacteria.

BACTERIA Groups: Spore forming and non-spore forming bacteria. Reproduction: Bacteria grow independently in the insect haemocoel*. Infection: Bacteria must be ingested. They rupture the gut wall, then invade the haemocoel. Survival: Some bacteria form spores which are moderately resistant. Host range: Lepidoptera, Coleoptera and Diptera. Culture: They can easily be grown in Petri dishes or in liquid culture BUT specific conditions are needed for spore formation in artificial media. Biocontrol agents: The main bacterial control agents of insects are species from the genus Bacillus. Other groups exist but typically only cause disease in weak, injured or stressed insects.

Bacillus spp. Bacillus thuringiensis: During sporulation the bacteria produce a large proteinaceous crystal which is bipyramidal in shape and also a thick walled endospore*. The crystal is an endotoxin* which dissolves inside the host in alkaline gut fluids and releases toxic polypeptides*. Mainly Lepidoptera are attacked, but strains specific to mosquitoes and Coleoptera are also known. Developed as a commercial product, widely available throughout the world. The Bt toxins have been widely used in the first generation of genetically modified crop plants. Bacillus popilliae: Produces no toxins in the infection cycle. Japanese beetle (Popillia japonica) larvae ingest spores in the soil which germinate and produce vegetative cells which fill the gut in three to five days. Some cells penetrate the gut wall, then grow and sporulate in the haemolymph. 14-21 days after initial infection the insect body is swollen and creamy white (milky disease). After death, the spores are released into the soil and establish persistent infection sites. Bacillus sphaericus: Certain strains produce a proteinaceous toxin which poisons mosquito larvae. Host death may also be induced by the spores alone.

Bacillus thuringiensis viewed by phase contrast microscopy Bacillus thuringiensis viewed by phase contrast microscopy. The vegetative cells contain endospores (phase bright) and crystals of an insecticidal protein toxin (delta endotoxin). milky spore disease Bacillus popilliae Dutky

Bt-enginered crops Since 1996, a wide range of crop plants have been genetically engineered to contain the delta-endotoxin gene from Bacillus thuringiensis. These "Bt crops" are now available commercially in the USA. They include "Bt corn", "Bt potato", "Bt cotton" and "Bt soybean". Such plants have been genetically engineered to express part of the active Cry toxin in their tissues, so they kill insects that feed on the crops. it ensures that only those insects that attack the crop will be exposed to Bt toxins - there is no risk to other types of insect. It also ensures that the range of uses for Bt is extended to insects that feed on the roots or that bore into the plant tissues - for example, the European corn borer - because such insects cannot be controlled by Bt suspensions sprayed onto plant surfaces. However, there is also a "downside", because the target insects are perpetually exposed to toxins and this creates a very strong selection pressure for the development of resistance to the toxins. Various crop-management strategies are being developed to try to minimise this risk.

FUNGI Groups: Entomopathogenic fungi are found in several subdivisions. The fungi in the Entomophthorales (e.g. Entomophaga grylli) have complex life cycles involving a sexual stage and resting spores, whereas Deuteromycete fungi (e.g. Metarhizium and Beauveria) have a simple life cycle with no known sexual stage. Reproduction: Fungi reproduce mainly by spores which are formed sexually or asexually. Infection: Spores germinate on the host cuticle and penetrate using enzymes and mechanical pressure. Inside the haemocoel the fungus multiplies rapidly by budding or hyphal fission. The resulting yeast-like cells (blastospores) spread through the body. Death of host: By extensive mycelial colonization, causing asphyxiation or starvation, or by toxins released in the yeast phase. The cadaver desiccates as the hyphae use host nutrients and water to develop. Survival: Hyphae break through the cuticle after death. Spores may be liberated passively or actively to continue the infection cycle.

MAJOR GROUPS OF ENTOMOPATOGENIC FUNGI Sub-divisions Orders Families Example Mastigomycotina Oomycetes Lagenidiaceae Lagenidium Chytridiomycetes Blastocladiaceae Coelomomyces Zygomycotina Entomophthorales Entomophthoraceae Entomophaga and many other genera Mucorales Mucoroceae Sporodiniella Ascomycotina Clavicipitales Clavicipitaceae Cordyceps Hypocreales Hypocreaceae Cordycepioideus Laboulbeniales Laboulbenioceae Many genera Pleosporales Podonectriaceae Podonectria Basidiomycotina Septobasidiales Septobasidiaceae Septobasidium Deuteromycotina (No formal classification) Hyphomycetes Verticillium Aspergillus Beauveria Metarhizium Coelomycetes   Sorosporella

Entomophthorales Reproduction: Entomophthoralean fungi have complex life cycles involving non-sexual conidia, and sexual resting spores. Culture: Entomophthorales fungi must be cultured in complex media and some cannot be cultured in artificial media. Some produce mycelium* but will not sporulate. Entomophaga maimaiga is a widespread gypsy moth pathogen in Asia and epizootics (massive reproduction of the pathogen causing widespread disease in its host) are frequent in Japan. Entomophaga praxibulli: has been introduced to the USA for control of grasshoppers. Zoophthora radicans: has been used as a classical biocontrol agent against aphids in Australia. Entomophthora muscae: a natural enemy of flies Neozygites floridana: used against Mononychellus tanajoa, cassava green mite, in Benin.

Overwintering resting spores (azygospores) of Entomophaga maimaiga. Conidia of Zoophthora radicans Neozygites sp. Sporangiophora and sporangium of Entomophthora

Deuteromycetes Reproduction: The deuteromycete fungi have no known sexual stage. Although they do have mechanisms for the exchange of genetic material, most reproduction is asexual, by the production of asexual spores called conidia. Culture: Many deuteromycetes are easily cultured in simple artificial media. Metarhizium: M. anisopliae, and the sub-group var. acridum have been produced on a large scale on rice substrate in several tropical countries. Beauveria: B. bassiana and B. brogniarti have been produced on a large scale in China, Europe and America. Verticillium lecanii: has been developed as a biopesticide for use in glass-houses in Europe.

Metarhizium anisopliae Metarhizium anisopliae, formerly known as Entomophthora anisopliae, is a widely distributed soil-inhabiting fungus. The first use of M. anisopliae as a microbial agent against insects was in 1879, when Elie Metchnikoff used it in experimental tests to control the wheat grain beetle, Anisoplia austriaca. It was later used to control the sugar beet curculio, Cleonus punctiventris. M. anisopliae is categorized as a green muscardine fungus due to the green color of the sporulating colonies. It has been reported to infect approximately 200 species of insects and other arthropods. M. anisopliae generally enters insects through spiracles and pores in the sense organs. Once inside the insect, the fungus produces a lateral extension of hyphae, which eventually proliferate and consume the internal contents of the insect. Hyphal growth continues until the insect is filled with mycelia. When the internal contents have been consumed, the fungus breaks through the cuticle and sporulates, which makes the insect appear "fuzzy." M. anisopliae can release spores (conidia) under low humidity conditions (<50%). In addition, M. anisopliae can obtain nutrition from the lipids on the cuticle. The fungus can also produce secondary metabolites, such as destruxin, which have insecticidal properties on moth and fly larvae.

Metarhizium anisopliae

Beauveria bassiana Spesies Beauveria yang banyak digunakan untuk pengendalian hayati adalah B. bassiana yang dikenal sebagai white muscardine fungus karena membentuk masa putih ketika bersporulasi. B. bassiana menyerang berbagai spesies serangga hama penting dalam ordo Orthoptera, Hemiptera, Coleoptera, Diptera, Lepidoptera, dan Hymenoptera. B. bassiana tumbuh cukup cepat, koloninya mencapai diameter 1-3 cm dalam waktu 7 hari pada medium PGA pada suhu 25oC. Koloninya menyerupai kapas dengan warna permukaan putih, putih kekuningan, atau jingga pucat dan warna dasar koloni putih atau pucat. Hifa bening, bersekat, dan sempit. Sel konidiogen cenderung membentuk gugus padat yang tampak sebagai bola bertepung di antara hifa udara bila dilihat dengan mikroskop. Sel konidiogen pada hifa berbentuk erlenmeyer dengan pembengkakan pada dasar dengan filamen tipis berkelok sebagai konidiofor pada ujungnya. Pada bagian lateral dari filamen (konidiofor) tersebut dibentuk konidia pada setiap titik keloknya menurut pola pertumbuhan sympodial geniculate dengan konidia termuda terletak paling ujung (acropetal). Konidia berukuran sangat kecil (2-4 µm), bening, bersel tunggal, berbentuk globose sampai ovoid.

Beauveria bassiana

Spicaria (obsolete) (=Paecilomyces) Nama ilmiah jamur yang sudah obsolete (tidak sesuai dengan kaidah taksonomi) Sekarang dinamakan Paecilomyces (sebagian besar), Beauveria, Namuraea, Isaria (misal: S. javanica menjadi P. javanica) Identifikasi didasarkan atas warna koloni dan sifat P. crustaceus dan P. variotii dapat tumbuh sampai suhu 50° dan bahkan 60°C. Koloni tumbuh cepat dan dewasa dalam 3 hari, rata, bertekstur tepung atau velvet, mula-mula putih dan kemudian menjadi kuning, kuning-hijau, kuning-cokelat, kuning jeruk-coklat, jingga, atau ungu tergantung pada spesies. Dari bawah cawan petri koloni berwarna putih kotor, buff, atau cokelat. Koloni tua mengeluarkan bau aromatik. Hifa bening bersekat, konidiofora (lebar 3-4 µm dan panjang 400-600 µm) umumnya bercabang dan di ujungnya terdapat fialida yang meruncing ke arah ujung, mengelompok dalam pasangan menyerupai sikat. Konidia bersel tunggal, bening sampai gelap, berpemukaan halus atau kasar, berbentuk oval atau fusoid, dan membentuk rantai panjang. Klamidospora jarang ditemukan. Teleomorf: Thermoascus (Ascomycotina)

Paecilomyces farinosus

Verticillium lecanii V. lecanii (=Cephalosporum lecanii) pertama kali dideskripsikan pada tahun 1861, merupakan fungi kosmopolitan yang menginfeksi serangga, khususnya kutu perisai pada kawasan tropika dan sub-tropika. Fungi ini dikenal sebagai fungi halo putih karena pertumbuhan miselia putih pada tepi koloni yang terbentuk pada serangga yang diinfeksi. Konidia lengket sehingga mudah menempel pada permukaan tubuh serangga. Konidia berkecambah pada permukaan tubuh serangga dan mempenetrasi integumen untuk tumbuh di dalam dan membinasakan bagian dalam tubuh serangga, kemudian tumbuh keluar tubuh dan bersporulasi di luar tubuh sehingga serangga terinfeksi tampak putih sampai putih kekuningan dalam waktu  7 hari pada suhu 15-25oC dan kelembaban nisbi 85-90% selama 10-12 jam. Miselium V. lecanii menghasilkan toksin siklodepsipeptida yang disebut basianolida yang dapat membunuh ulat sutera dan toksin lainnya misalnya asam dipikolinat. Aktivitas V. lecanii tergantung pada strain-nya, yang membentuk konidia kecil menginfeksi Aphis spp., yang membentuk spora agak besar menginfeksi lalat putih, dan strain lainnya dapat menginfeksi jamur karat. V. lecanii telah diproduksi secara komersial untuk mengendalikan Myzus persicae dan Aphis gossypii.

VIRUSES Reproduction: Viruses multiply by independent synthesis of their component parts. These parts assemble to produce progeny virus within the host cell. Infection: Viruses normally enter the host through the mouth. The protein coat of the virus dissolves in the gut, releasing the virus particles (virions*). The virions invade, then multiply in the cells of the gut wall. Replication on a massive scale then takes place in the fat body, haemocytes* and hypodermis*. Death of host: Death usually occurs in three to ten days. Survival: After death the body ruptures and releases millions of occlusion bodies. Occlusion bodies protect the virus which can persist for years in the right conditions. Host range: The host range is narrow, but viruses are known from a wide range of hosts. Culture: Viruses can only be cultured in the live insect host or in tissue culture. Groups: Baculovirus, entomopox virus, picornavirus, cytoplasmic polyhedrosis virus. Biocontrol agents: At present, the main group of viruses used as biological control agents are the baculoviruses (see below).

Figure 1. Generalized life-cycle of insect viruses Figure 1. Generalized life-cycle of insect viruses. Figure credit: Jim McNeil, Department of Entomology, Penn State University.

Viruses Used for Biocontrol COMMODITY INSECT PEST VIRUS USED PRODUCT Apple, pear, walnut and plum Codling moth Codling moth granulosis virus Cyd-X 3) Cabbage, tomatoes, cotton, (and see pests in next column) Cabbage moth, American bollworm, diamondback moth, potato tuber moth, and grape berry moth Cabbage army worm nuclear polyhedrosis virus Mamestrin* 5) Cotton, corn, tomatoes Spodoptera littoralis Spodoptera littoralis nuclear polyhedrosis virus Spodopterin* (5) Cotton and vegetables Tobacco budworm Helicoverpa zea, and Cotton bollworm Heliothis virescens Helicoverpa zea nuclear polyhedrosis virus Gemstar LC, Biotrol, Elcar (3) Vegetable crops, greenhouse flowers Beet armyworm (Spodoptera exigua) Spodoptera exigua nuclear polyhedrosis virus Spod-X (3) Vegetables Celery looper (Anagrapha falcifera) Anagrapha falcifera nuclear polyhedrosis virus none at present Alfalfa and other crops Alfalfa looper (Autographa californica) Autographa californica nuclear polyhedrosis virus Gusano Biological Pesticide (3) Forest Habitat, Lumber Douglas fir tussock moth (Orgyia psuedotsugata) Orgyia psuedotsugata nuclear polyhedrosis virus TM Biocontrol (2) Gypsy moth Lymantria dispar) Lymantria dispar nuclear polyhedrosis virus Gypchek

Baculoviruses Description: Baculoviridae are the safest insect viruses to use as pathogens, since no similar viruses are known to infect vertebrates or plants. They have double-stranded DNA and are protected by a protein coat which improves their persistence. Infection: Infection occurs after susceptible insect larvae eat food contaminated with virus. The virus then attacks the haemolymph, fatty tissue and mid gut. The insect becomes paralysed. Virulence: Highly virulent; the presence of very few particles can initiate infections and hosts die within 3-10 days. Susceptibility: The gut of the host insect must be alkaline so that the occlusion body can dissolve. Locusts: No baculovirus has been recorded from locusts with the exception of an unconfirmed report of cross infection from a Spodoptera sp. (Lepidoptera) host. Groups: Three groups of baculoviruses are described below: the nuclear polyhedrosis viruses, granulosis viruses and group C baculoviruses.

Rod-shaped virions are enveloped in the cell nucleus and occluded by a protein matrix The occlusion bodies are highly resistant and can survive long periods in the environment

Baculoviruses: Nuclear polyhedrosis viruses (NPV Description: About 280 species known. Rounded cubic or hexagonal polyhedra. 0.5-1.5 microns (µm) in size. Singly or multiply envelopped (see Figure 1). Infection: Infection occurs in the adipose* tissue of the hypodermis, in the tracheae and the middle intestine. Host: Approximately 120 species of Lepidoptera and Hymenoptera (particularly saw-flies). Each virus is highly specific to its host. Survival: Nuclear polyhedrosis viruses form particles inside a crystalline protein structure (occlusion body*). This allows the virus to survive outside the host for years out of sunlight. Biocontrol agents: The following NPVs have all been produced on a commercial or semi-commercial scale: Autographa californica NPV, Lymantria dispar NPV, Malacasoma disstria NPV, Mamestra brassicae NPV, Neodiprion sertifer NPV, Spodoptera NPV and Heliothis NPV

Baculoviruses: Granulosis viruses (GV) Description: There are about 65 species of granulosis virus, with oval or ovoid granules. Infection: Granulosis viruses attack the adipose* tissue. Host: Lepidoptera larvae. Survival: Granulosis viruses form particles inside a crystalline protein structure (occlusion body*). This allows the virus to survive outside the host. Can survive for years out of sunlight. Biocontrol agents: Include Cydia pomonella GV (codling moth), Phthorimaea operculella GV (potato tuber moth).

Baculoviruses: Group C Baculoviruses NB This group of viruses is currently unclassified Description: Double stranded DNA. Viruses with non-included virions. Only visible using an electron microscope. 22-30 nm in size. These viruses are unusual since they have no protective protein coat to help them to survive. Infection: These viruses attack the haemolymph*, fat body, mid-gut. Insects become paralysed. Host: These viruses are restricted to Arthropoda. Larvae and adults of Coleoptera, Hymenoptera and mites. Biocontrol agents: Baculovirus oryctes: Used for the control of rhinoceros beetles, Oryctes spp. This virus is excreted from the living diseased insect as virions*. These are passed on to other adults during mating. Some spread occurs from contamination of adult breeding and larval feeding sites, but the virus does not survive long in the environment.

Entomopox viruses Description: Entomopox viruses have inclusion bodies (i.e. they are occluded* viruses) which are important for identification. Spherical or ovoid particles. 5-20 µm in size. Infection: These viruses must be ingested by the host. They then attack the fat body. Virulence: They kill hosts more slowly than baculoviruses. Host: Lepidoptera larvae, Diptera, Coleoptera, Orthoptera. Survival: Little is known. Locusts: Entomopox viruses have been recorded from locusts and grasshoppers. Further information: http://life.anu.edu.au/viruses/welcome.htm http://www.virology.net/

PROTOZOA Groups: Ciliophora, Sarcomastigophora, Apicomplexa, Microspora. Reproduction: Reproduction is asexual and occurs in the gut or fat body cells by multiple or binary fission. The fusion of two gametes* forms a zygote* which divides repeatedly. Host range: Wide range in insects, mammals and man but especially obligate pathogens of arthropods. Death of host: Very slow. Not highly pathogenic but they do reduce the rate of development and fecundity. Infection: Chronic rather than lethal caused by ingesting spores followed by penetration of digestive tract. Spores have a polar capsule which after ingestion and germination develops into a tube which can penetrate gut cell walls. Protozoa kill the host only at very high levels. Survival: The liberated spores are highly resistant. Transmission of spores is also possible through the eggs. Culture: In live insects only.

PROTOZOA AS Biocontrol agents: Malamoeba : Malamoeba locustae mainly known as a contaminant of laboratory cultures of locusts. Microspora: The most important protozoans for biological control. Nosema: There are some species which can be used in biocontrol. Nosema locustae infects a wide range of grasshoppers. Vairimorpha: Vairimorpha necatrixis a broad spectrum agent which is infectious to many Lepidoptera.

Electron micrograph showing the ultrastructure of a spore of Nosema locustae. A anchoring disc, PF polar filament, PP polaroplast, S sporoplasm, EN endospore, EX exospore, (magn. ca. 20,300 x). In phase contrast, especially light refracting cysts are characteristic diagnostic stages of this pathogen, isolated from Malpighian tubules of Locusta migratoria, (phase contrast, magn. ca. 870x) Giemsa-stained smear of spores (S) and sporoblasts (Sb) of Nosema locustae (bright field, magn. ca. 1540 x). Electron micrograph showing the ultrastructure of a spore of Nosema locustae. A anchoring disc, PF polar filament, PP polaroplast, S sporoplasm, EN endospore, EX exospore, (magn. ca. 20,300 x) Extruded polar filament of a spore of Nosema locustae, (phase contrast, magn. ca. 970 x).

NEMATODES Groups: There are eight or nine orders associated with insects. Most biocontrol agents are among the Mermithidae and Senecentia. Reproduction: Adults are bisexual. They develop through four larval moults before reaching maturity. Survival: Generally grow in the host and develop in the environment, in both aquatic and terrestrial habitats.

Mermithidae Subclass Penetrantia, family Mermithidae; a large group of obligate parasites. Infection: These invade through the cuticle at the second larval stage and develop to the fourth larval stage filling the host body cavity. This shows as a swollen abdomen. Death of host: Larvae emerge after rupturing the host cuticle. Survival: Mature in surrounding soil and water Culture: In live insects or in macerated liver. Host range: Romanomermis culicivorax can kill early instar mosquito larvae. Other mermithids sometimes attack locusts and grasshoppers.

Steinernema carpocapsae

Secernentia These are free living saprophytes and do not kill insects. Exceptions are from the genera Steinernema (=Neoaplectana) and Heterorhabditis, known as rhabditid nematodes. Infection: Free-living third instar rhabditid larvae are attracted chemically to susceptible hosts. They penetrate through the mouth or anus, enter the haemocoel* via the gut wall. Some may enter directly through the cuticle. They release highly virulent bacteria (Xenorhabdus spp.) into the haemocoel. Death of host: From septicaemia in 2-10 days. Survival: Nematodes feed and develop on host tissues and bacteria. Third stage infective larvae containing bacterial inoculum emerge from the cadaver 10 days after penetration. The larvae need free water to disperse in. Culture: In live insects or in macerated liver.

Heterorhabditis bacteriophora Insect-pathogenic nematodes of both genera Heterorhabditis and Steinernema have independently entered a symbiotic relationship with bacteria. The bacteria belong to the family of Enterobactericeae, and are hence closely related to Escherichia coli, the probably best studied bacterium which lives in our intestine. The Nematode symbionts, however, are not harmful for humans and they can not grow at temperatures of more than 35°C. It is major advantage of this symbiosis, that the insects entered by the Nematode dyes relatively quick. There are a number of closely related nematodes without symbiotic bactreria, that do also enter insectsbut that subsequently have to wait until the insect dyes for other reasons. Et other species only enter dead insect carcasses. By carrying the symbiotic bacteria, these two genera have conquered a new ecological niche and a new food source. The same holds true for the bacteria. Most of the species can not kill the insects unless they are carried into the insect's haemocoel.