昆虫病原真菌紫外线诱变育种研究进展

昆虫病原真菌是控制害虫发生与危害的重要生物防治因子之一,优良的菌种是生物防治的关键。紫外线诱变育种是昆虫病原真菌菌种选育中最常用的方法之一,具有易操作、效果好、作用时间长等优点。本文以白僵菌、绿僵菌、玫烟色棒束孢等三种常用的昆虫病原真菌为例,从诱变育种的材料、方法和应用等方面综述了昆虫病原真菌紫外线诱变育种的研究现状,以期为昆虫病原真菌的生物防治研究提供参考。     

The secretome and chemistry of Metarhizium; a genus of entomopathogenic fungi

The major fungal phyla have independently and repeatedly evolved the ability to overcome the insect defense system, which is usually highly effective against fungal attack. During the genomic era of the last decade, extensive progress has been made in understanding the mechanisms of interactions between insects and their fungal pathogens, particularly Metarhizium spp., the focus of this article. New models of pathogenesis have been designed, new biological phenomena have been discovered, and a plethora of new molecules and functions have been determined that are crucial for successful establishment of fungal disease by permitting: attachment to the insect surface; germination and formation of infection structures; penetration of the host; colonization of host tissue; and sporulation on cadavers. Genomic technologies in particular are producing breakthroughs in some of the more intractable aspects of this field, e.g. evolution of host specificity.     

Virulence of entomopathogenic fungi against Culex pipiens: Impact on biomolecules availability and life table parameters

Culex pipiens mosquitoes considered as vectors for many arboviruses such as the West Nile virus and encephalitis virus showing a global impact on human health. The natural management of the aquatic stages of this pest is crucial for maintaining an insecticide-free and sustained environment. The present work focused on studying the biological and biochemical effects of the entomopathogenic fungi: Metarhizium anisopliae, Beauveria bassiana, and Paecilomyces lilicanus, against 3^rd instar larvae of Culex pipiens laboratory colony. The results revealed that M. anisopliae showed maximum larval mortality (88%) with the lowest lethal time (LT₅₀) (22.6 hrs) at 10⁸ spores/ml followed by B. bassiana (73.33%) with LT₅₀ (38.35 hrs), while P. lilicanus showed minimum percent mortality (65%) with highest LT₅₀ (51.5 hrs). The median lethal concentration (LC₅₀) values were found to be 1.027 × 10⁵ spores/ml for M. anisopliae, 1.24 × 10⁶ spores/ml for B. bassiana, while it was 8.453 × 10⁶ spores/ml for P. lilicanus. A reduction in female fecundity, number of hatched eggs, pupation and adult emergence percentage were recorded. The biochemical analysis of the treated larvae revealed different quantitative decrease in total soluble proteins, lipids, and carbohydrate hydrolyzing enzymes compared to control. Histopathological effects of fungal infection upon insect cuticles, muscles, and midgut were investigated. Based on the obtained results, M. anisopliae proved its superior virulent effect as a bio-control agent against Cx. pipiens.     

Use of quantitative PCR to understand within-host competition between two entomopathogenic fungi

Interactions between the entomopathogenic fungi Zoophthora radicans and Pandora blunckii infecting larvae of Plutella xylostella were investigated. This is the first report to quantify within-host growth of one fungus in the presence of another competing for the same host resource using quantitative PCR (qPCR) at regular time points during the infection process. In larvae inoculated only with Z. radicans, there was a cumulative increase in the quantity of Z. radicans DNA throughout the time course of infection. However, in dual-inoculated larvae, there was an initial accelerated rate of growth of Z. radicans compared to when it was inoculated alone, but by the time of host death it had been effectively excluded by P. blunckii. The implications of these results for co-existence of these fungal pathogens in the field are discussed.     

Dual biocontrol potential of the entomopathogenic fungus Akanthomyces muscarius against Thaumetopoea pityocampa and plant pathogenic fungi

Akanthomyces spp. species are known for their capacity to biocontrol of certain insects and plant pathogens; however, their ability to biocontrol the pine processionary (Thaumetopoea pityocampa) and certain phytopathogenic fungi belonging to the genera Fusarium and Curvularia have not been studied before. In this study, a strain from Akanthomyces muscarius was isolated from wheat grains and then identified by morphological and molecular tests. The strain was further studied for its capacity to control Thaumetopoea pityocampa larvae through dose-mortality tests, and its ability to control some phytopathogenic fungi strains of the genera Fusarium and Curvularia was studied through direct confrontation tests. Dose-mortality tests at three concentrations of Akanthomyces muscarius against the first instar larvae revealed a mortality of 92.15% after 11 days for the concentration of 2.3 × 10⁶ conidia.ml⁻¹, with a median lethal concentration of 7.6 x10³ conidia.ml^1. Our isolate also showed antifungal activity against these phytopathogenic fungi with inhibition rates ranging from 39.61% to 52.94%. Akanthomyces muscarius proved to be a promising biocontrol agent for plant pests and diseases.     

虫生真菌的分子生物学检测技术

线粒体DNA探针、核糖体RNA编码DNA序列分析、随机扩增多态性DNA－聚合酶链式反应、基因组DNA探针和电泳核的,五类分子生物学技术在虫生真菌研究中已有应用。本对此作了介绍,着重阐述了RAPD－PCR技术的特点及其使用此技术的研究成果。     

Integration of entomopathogenic fungi and eco-friendly insecticides for management of red palm weevil,Rhynchophorus ferrugineus (Olivier)

Red palm weevil (Rhynchophorus ferrugineus) is a voracious pest of date palm worldwide. Pakistan ranks sixth in date palm production globally. Losses to date palm plantations in Pakistan sometimes surpass 10%-20%. Most of the traditional management strategies used by farmers have been found insignificant to combat this voracious pest. The entomopathogenic fungi, Beauveria bassiana [QA-3(L) and QA-3(H)] and insecticides, Nitenpyram (Active 10% SL) [NIT (L) and NIT (H)] were applied to larval (2nd, 4th, and 6th), pupal and adult stages of R. ferrugienus. Integration or alone application of fungi with insecticides at different concentration under laboratory conditions. Combined application was depicted additive and synergistic interactions. Contrarily, highest cumulative mortality (100%) was recorded in 2nd instar larvae as compared to later instar larvae at combined application. The maximum pupal and adult mortality remained 89% and 66% respectively after treatment with [QA-3 (H) + NIT (L)]. The combination of B. bassiana at higher concentration whereas Nitenpyram at lower dose was found more lethal to larvae, pupae and adults of R. ferrugineus. This signifies the need of combining B. bassiana and bio-rational insecticides that can reduce the cost of management with least harm to environment and natural enemies.     

Influence of commercial fungicides on the germination,growth and virulence of four species of entomopathogenic fungi

The influence of 15 commercially available fungicides on the germination, growth and virulence of Metarhizium anisopliae, Beauveria bassiana, Isaria fumosorosea, and Lecanillium longisporum was evaluated. The influence of the fungicides on conidial germination was dependant on the fungicide type and dose. Most fungicides retarded conidial germination of all the fungi tested at 10× and at the recommended rate of application, however, their toxicity declined at lower concentrations. Most of the fungicides inhibited mycelial growth of B. bassiana, whereas L. longisporum growth was unaffected. Only two and eight fungicides influenced mycelial growth of I. fumosorosea and M. anisopliae, respectively. None of the fungicides influenced the virulence of B. bassiana and L. longisporum, however, tolylfluanid and azoxystrobin reduced the virulence of M. anisopliae and I. fumosorosea, respectively. These studies clearly show that certain fungicides have the potential to inhibit germination of entomopathogenic fungi in vitro but appear to have little or no effect on their virulence against target insects.     

Control of Bemisia tabaci by entomopathogenic fungi isolated from arid soils in Argentina

Entomopathogenic Hypocreales were isolated from arid soils in Argentina using Tenebrio molitor as bait and tested for their biological performance at 30°C and 45–65% RH. Conidial germination was tested in three vegetable oils (sunflower, olive and maize) at two concentrations (1% and 10%) to evaluate their compatibility for further liquid formulations. According to radial growth and germination results, we selected four isolates to test their pathogenicity against second instar B. tabaci nymphs with the selected oil formulations at 30°C. CEP381 and CEP401 showed the highest radial growth. Isolates CEP381, CEP401, CEP413 and CEP409 (Metarhizium spp.) had similar germination percentages as compared with water control when germinated on either sunflower, olive or maize oils at 10% v/v. The highest mortality of B. tabaci was observed for the isolates CEP381 in sunflower oil and CEP401 in olive oil. Molecular identification of isolates was performed using ITS4–5 primers. All isolates belong to the Metarhizium core group. Tested isolates could grow and infect B. tabaci nymphs at 30°C in some of the vegetable oils as carriers, providing new possibilities for integrated pest management of Bemisia tabaci.     

Influence of plant resistance at the third trophic level: interactions between parasitoids and entomopathogenic fungi of cereal aphids

Host-plant resistance can affect herbivorous insects and their natural enemies such as parasitoids and entomopathogenic fungi. This tritrophic effect acts on interspecific interactions between the two groups of natural enemies distantly related in phylogenetic terms. The intra- and extra-host aspects of the interaction between the cereal aphid parasitoid Aphidius rhopalosiphi and the entomopathogenic fungus Erynia neoaphidis developing on the grain aphid, Sitobion avenae, on resistant and susceptible wheat (Triticum aestivum) cultivars, were studied. The competitive outcome of the intra-host interaction depended on the timing of parasitoid oviposition and fungal infection and was affected by wheat resistance. In particular, survival of the parasitoid was lower on the resistant wheat cultivar than the susceptible wheat cultivar, when the competitive outcome of the interaction was favourable for either parasitoid or fungal development. Before and after this period the influence of plant resistance was not significant. Furthermore, the extra-host interaction was not affected by the wheat cultivar, although an increase in fungal infection of S. avenae was observed when parasitoids foraged in the experimental arena with sporulating aphid cadavers compared with foraging in the absence of sporulating cadavers. Our results showed that the host plant may affect interspecific interactions between parasitoids and fungi and that these interactions depended on the timing of parasitoid oviposition and fungal infection. Received: 16 March 1998 / Accepted: 24 August 1998     

Temporal and spatial gradients of humidity shape the occurrence and the behavioral manipulation of ants infected by entomopathogenic fungi in Central Amazon

Some species of the fungal genus Ophiocordyceps that parasitize ants have evolved some degree of behavioral manipulation to increase their own transmission. Carpenter ants (Camponotus spp.) infected by Ophiocordyceps unilateralis species climb vegetation and bite leaves or other plant parts before death, while other ants infected by other Ophiocordyceps species die buried within or on the leaf-litter, attached to stems and tree trunks. The microenvironment of the host's death location is important for fungal development and dispersal to new hosts, being an interesting example of an extended phenotype. This study investigated the effect of humidity variation in space (15 km²) and time (14 months) on the occurrence and expression of the extended Ophiocordyceps phenotype parasitizing ants in Central Amazonia. We found O. unilateralis s.l., O. kniphofioides s.l. and O. australis s.l. parasitizing 30 ant species, but O. unilateralis s.l. was by far the most abundant fungus. Parasitized Camponotus atriceps and C. bispinosus were more abundant and died in higher places in periods with greater air humidity. Otherwise, the abundance and height of C. senex cadavers were higher in drier plots. These results provide insights on how the spatial and temporal variation in air humidity may shape the occurrence and behavioral manipulation of ants infected by entomopathogenic fungi at larger scales.     

Occurrence of entomopathogenic fungi in migratory alate aphids in Yunnan Province of China

Occurrence of entomopathogenic fungi in migratory alate aphids was investigated by trapping air-borne aphids on cabbage and wheat plants and observing the insects for fungal infection. Potted cabbage and wheat plants put 30 m apart on yellow cloth, were placed on the top of a tall building (six-story building) in Yunnan Province, China away from cultivated fields or other vegetation. Seven species of aphids trapped on the plants collected and monitored for fungal infection in the laboratory at a daily basis from 19 February to 31 December, 2003. Aphids were trapped every day during the experimental period. A total of 3,681 aphids were trapped on cabbage and 3,028 on wheat. Myzus persicae (Sulzer), Brevicoryne brassicae (L.), and Sitobion avenae (F.) were the major aphid species accounting for 94.7 and 93.1% of the insect specimens landing on cabbage and wheat, respectively. Other aphis species encountered included, Acyrthosiphon pisum (Harris), Lipaphis erysimi (Kaltenbach), Pemphigus sinobursaris Zhang, and Rhopalosiphum padi (L.). Preference for host plant played significant role in attraction of aphids, especially of the three major species. Accumulated over the whole experimental period nearly 33% of the aphids landing on cabbage and 37% landing on wheat were infected with fungi. Erynia neoaphidis Remaudiere and Hennebert, Entomophthora planchoniana Cornu, and Beauveria bassiana (Balsamo) Vuilemin were the major fungal pathogens encountered and accounted for nearly 95% of the infested aphids on cabbage and 97% on wheat. Other entomopathogenic fungi encountered included Zoophthora radicans (Brefeld) Batko, Conidiobolus obscurus (Hall and Dunn) Remaudiere, and Neozygites fresenii (Nowakowski), Paecilomyces fumosoroseus (Wize) Brown and Smith, and Aspergillus flavus Link. Although the number of aphids trapped throughout the experimental period did not vary consistently with temperature or humidity, the fungal was directly related to relative humidity. Higher insect mortality, especially due to Entomophthorales, positively related relative humidity.     

Potential of a combination of entomopathogenic fungal strains and a non-ionic surfactant to control the fall armyworm (Spodoptera frugiperda)

The fall armyworm, Spodoptera frugiperda, is an emerging invasive pest in Taiwan that feeds on a wide range of crops and causes serious damage. Herein, an entomopathogenic fungal library (EFLib) was constructed to identify potential microbes to control FAW. Twenty-eight indigenous entomopathogenic fungi (EPF) were isolated and investigated for their potential pathogenicity, with Metarhizium pinghaense (Mp-NCHU-124) and Beauveria bassiana (Bb-NCHU-157) exerting dose-dependent effects on the 4th instar FAW larvae. The non-ionic surfactant Silwet L-77 rapidly killed FAW larvae after spraying at a concentration of 300 mg/kg and the toxic effect of Silwet L-77 on FAW larvae was dose-dependent. When the EPF isolates (10⁶ conidia/mL) were applied to FAW larvae in combination with the non-ionic surfactant Silwet L-77 (30–90 mg/kg), the mortality rate dramatically increased and the LT₅₀ reduced, with increased fungal mycosis (Mp-NCHU-124: 38% to 72% and Bb-NCHU-157: 20 to 62%), indicating the high compatibility of EPF with the non-ionic surfactant. Thus, the Silwet L-77+EPF combined formulation has potential for practical field application for FAW pest control and sustainable agriculture in the future.     

Biochemical analysis and pathogenicity of entomopathogenic fungi to Diaphorina citri Kuwayama (Hemiptera: Liviidae)

Asiatic citrus psylla, Diaphorina citri, Kuwayama (Hemiptera: Liviidae) is an economic pest of citrus groves and a vector of the bacterium, Candidatus Liberibacter spp., one of the causative agents of citrus greening. In order to estimate the infectivity of six different isolates of Beauveria bassiana, Metarhizium anisopliae and Isaria fumosorosea, fungal bioassay was performed on the adults of D. citri. Adults of D. citri were treated individually with 1 × 10⁵, 1 × 10⁶, 1 × 10⁷, 1 × 10⁸, 2 × 10⁸ spores/mL fungal concentrations by the immersion method. Subsequent to fungal bioassay, treated D. citri were used to determine the levels of esterase and glutathione S‐transferases (GST) enzymes over a period of 3–7 days. The mortality results suggest that I. fumosorosea isolates (If‐02) caused 82.2% mortality on the seventh day of treatment. However, B. bassiana isolate (Bb‐08) with lowest LC₅₀ (1.4 × 10⁷ spores/mL) proved to be highest potential isolate against D. citri. Biochemical determination of esterase and GST activity assay showed significant differences in activities after infection of fungi. Significantly high activity of esterase was observed by Bb‐01 (27.0 unit per mg protein) on the seventh day, while Ma‐11.1 and If‐2.3 (16.9 and 36.3 unit per mg protein) on the third day post treatment. However, maximum GST's activity was showen by isolates Bb‐08,Ma‐M₂ and If‐2.3 (37.6, 1.40 and 10.9 unit per mg protein) on the third day. The current investigation will help to explore the relations between the insect defense system and entomopathogenic fungi. Moreover, the determination of enzymatic activities will be useful for selecting the most pathogenic isolates.     

Culture degeneration in conidia of Beauveria bassiana and virulence determinants by proteomics

The quality of Beauveria bassiana conidia directly affects the virulence against insects. In this study, continuous subculturing of B. bassiana on both rice grains and potato dextrose agar (PDA) resulted in 55 and 49 % conidial yield reduction after 12 passages and 68 and 60 % virulence reduction after 20 and 12 passages at four d post-inoculation, respectively. The passage through Tenebrio molitor and Spodoptera exigua restored the virulence of rice and PDA subcultures, respectively. To explore the molecular mechanisms underlying the conidial quality and the decline of virulence after multiple subculturing, we investigated the conidial proteomic changes. Successive subculturing markedly increased the protein levels in oxidative stress response, autophagy, amino acid homeostasis, and apoptosis, but decreased the protein levels in DNA repair, ribosome biogenesis, energy metabolism, and virulence. The nitro blue tetrazolium assay verified that the late subculture's colony and conidia had a higher oxidative stress level than the early subculture. A 2A-type protein phosphatase and a Pleckstrin homology domain protein Slm1, effector proteins of the target of rapamycin (TOR) complex 1 and 2, respectively, were dramatically increased in the late subculture. These results suggest that TOR signalling might be associated with ageing in B. bassiana late subculture, in turn affecting its physiological characteristics and virulence.     

Characterization in biological traits of entomopathogenic nematodes isolated from North China

In a series of bioassays, thirty-one isolates that were collected from diverse locations in northern China and the laboratory kept isolate Steinernema carpocapsae All, were compared in order to select superior isolates for biological control of Bradysia odoriphaga. Virulence of the isolates against B. odoriphaga was significantly different among nematode isolates. Tolerance of infective juveniles (IJs) to heat, cold, and desiccation differed significantly among and within species. Strains from S. carpocapsae, S. ceratophorum, S. longicaudum, Heterorhabditis indica, and H. bacteriophora were more heat tolerant than strains from S. feltiae, S. hebeiense, S. monticolum, and H. megidis. Heterorhabditis megidis, H. bacteriophora, and S. carpocapsae showed better cold tolerance than the other species. High desiccation tolerance was recorded for S. carpocapsae, S. hebeiense, and S. ceratophorum. The infectivity of IJ of these species against Galleria mellonella larvae was not significantly different between the treated and non-treated IJ after the nematodes had been exposed to 40 °C for 2 h, −5 °C for 8 h or 25% glycerin for 72 h. Nematode survival was significantly affected by exposure time and IJ concentration when exposed to 40 °C or −5 °C. All nematode isolates lost their infectivity against G. mellonella after exposure to −5 °C for 16 h, except for H. megidis LFS10, which had a low infectivity of 3.3%. A hierarchical classification analysis classified the isolates in four main clusters. The fourth cluster, composed of 13 isolates, grouped the isolates that scored well for most traits.     

被毛孢属两个昆虫病原真菌的重新描述

【目的】调查研究被毛孢属真菌资源,丰富该属的物种多样性,完善已知种在公共数据库中的分类信息。【方法】从贵州省发现两个分别寄生于同翅目沫蝉、鳞翅目松梢螟成虫的真菌标本GZUIFR-dj14和GZUIFR-dy1,通过形态学观察和基于tef1、ITS和28S r DNA 3个基因序列的分子生物学系统发育分析对其进行鉴定。【结果】两个病原真菌被鉴定为两个已知种,分别为长白山被毛孢(Hirsutella changbeisanensis)和雷州被毛孢(Hirsutella leizhouensis)。【结论】重新描述了这两种被毛孢的标本和无性型分离菌株,对其新寄主、新栖息地进行了报道,并补充了分子序列、系统发育分析等分类信息。     

Cuticle-degrading proteases of entomopathogenic fungi: from biochemistry to biological performance

Abstract

Entomopathogenic fungi are among the most successful biocontrol agents for preventing economic loss from insects. The identification of virulent species or isolates, the development of formulation technology and the improvement of efficiency are avenues being pursuing by researchers in diverse scientific disciplines. A successful entomopathogenic fungus deploys a combination of mechanical and biochemical processes to overcome the first defensive barrier in insects, the integument. A precise understanding of the mechanisms underlying fungal pathogenicity, particularly the roles of enzymes such as proteases, is essential in order to highlight the potential of entomopathogenic fungi and increase their virulence via genetic modifications. Cuticle-degrading proteases are divided into subtilisin-like (Pr1) and trypsin-like (Pr2) proteases, which are secreted in the initial stages of penetration. The biochemical structure contains the catalytic triad Asp39, His69 and Ser224 in addition to Ca²⁺ binding sites. Studies have shown a molecular weight of almost 19–47?kDa, an optimal pH of 7–12 and an optimal temperature of 35–45?°C. Different species or isolates of entomopathogenic fungi exhibit differences in the secretion and activity of cuticle-degrading proteases, which may indicate their virulence capacity. Genetic engineering techniques have been developed to create isolates with protease overexpression. Such isolates have significantly higher virulence against the host because they not only ensure fungal penetration but also exhibit direct toxicity to insects.