Effect of Beauveria bassiana and Metarhizium anisopliae (Deuteromycetes) upon the coffee berry borer (Coleoptera: Scolytidae) under field conditions

The effect of three strains of the fungus Beauveria bassiana (Balsamo) Vuillemin and two strains of Metarhizium anisopliae (Metschnikoff) Sorokin upon the coffee berry borer, Hypothenemus hampei (Ferrari), was studied in three coffee farms at different altitudes (450-1,100 m above sea level) in Soconusco, Chiapas, Mexico. The maximum average percentage mycosis varied according to altitude. At 450 m asl (El Rincon) mycosis was 14.3% for B. bassiana and 6.3% for M. anisopliae; at 880 m asl (Santa Anita) mycosis was 40.6% for B. bassiana and 12.6% for M. anisopliae, and at 1,100 m asl (Alpujarras) 33.9% for B. bassiana and 22. 1% for M. anisopliae. The effect of fungal mycosis through time was not significant (P > 0.01) in any of the farms, but there was a significant difference between the strains of the fungus (P < 0.01); the best strains being Bb25 and Ma4 at the lower altitude, Bb26 and Ma4 for the middle altitude and Bb26 and Ma4 at the higher altitude. Environmental factors such as temperature, relative humidity and rain were not correlated with the percentage mycosis caused by B. bassiana and M. anisopliae. However, in the case of B. bassiana there was a significant, positive correlation (P < 0.01) between the infestation levels of the pest and the mycosis response of the entomopathogen.     

外来入侵杂草的生物防治及生防因子对本地非目标种的影响

随着生物入侵所引起的生态及经济问题日益严重,对有害入侵生物的防治问题也备受人们关注。生物防治因具有持续、高效、安全等优点,已成为防治有害入侵生物的重要方法。传统生防是防治有害入侵杂草的一种重要方法。在简单介绍生物防治的基础上,重点阐述了传统生物防治的理论基础——天敌逃逸假说,生防因子对外来人侵种的影响及其对本地非目标种的直接和间接效应,并针对这些问题,对我国开展生物防治工作提出几点建议。     

Populational parameters of Spalangia endius Walker (Hymenoptera: Pteromalidae) on Pupae of Musca domestica L. (Diptera: Muscidae) treated with two strains of Beauveria bassiana (Bals.) Vuil. (Deuteromycetes)

The parasitoid Spalangia endius Walker is an efficient controller of Dipteran pupae, such as Musca domestica L. The entomopathogenic fungus Beauveria bassiana (Bals.) Vuil. is a regulator of insect populations, including these synanthropic pests. The aim of this work was to explore the possibilities of utilizing both agents in a combined form for the biocontrol of the domestic fly. Recently formed M. domestica pupae were inoculated by immersion in conidia suspension (10(8) conidia/ml) with two strains of B. bassiana (Bb6 and Bb10). The inoculated pupae were offered to the female parasitoid. In one bioassay they were offered pupae inoculated a single day and in other, pupae inoculated the following day as well. In both bioassays non inoculated (control) pupae were offered to the parasitoids until their death. Thirty females of S. endius were used for each strain and bioassay. From the study of the parasitoid offspring, life tables were built and the reproduction net rate (R(0)) and intrinsic natural increase (r(m)) were obtained among other demographic parameters; the parasitism percentages and sex ratios were also analyzed. B. bassiana did not affect significantly the biodemography of the parasitoid when pupae were inoculated a single time. On the other hand the R0 and the rm were smaller than that of the control without the fungus when pupae were inoculated twice, although sporulation was not observed in the cadavers of S. endius.     

Vegetative compatibility groups in indigenous and mass-released strains of the entomopathogenic fungus Beauveria bassiana: likelihood of recombination in the field

Using nitrate non-utilizing (nit) mutants, we determined vegetative compatibility groups (VCG) among strains of Beauveria bassiana representing strains indigenous to North America, isolated from diverse insect hosts, and strains that have been mass released for insect control. Genetic similarity among these strains was analyzed using random amplified polymorphic DNA (RAPD) markers. Our data revealed 23 VCGs among the 34 strains tested, with most of these groups comprised of only a single strain. We also observed a VCG comprised of eight genetically similar strains isolated from Colorado potato beetles (CPB). Co-inoculation studies of CPB larvae with complementary nit mutants from the same or from different VCGs revealed heterokaryosis in four out of five same-VCG pairs, with only 5-17% of the sporulating cadavers generating few parasexual recombinants. In contrast, none of the infected beetles treated with non-compatible pairs generated recombinants. The large number of VCGs observed and the low frequency of in vivo recombination limited to vegetatively compatible strains indicate that this self/non-self recognition system may be an effective barrier preventing genetic exchange between dissimilar strains in the field.     

Genetic groups of the insect-pathogenic fungus Beauveria bassiana are associated with habitat and thermal growth preferences

A persistent paradigm in insect pathology is one that relates the insect host to certain genetic groups of insect-pathogenic fungi. This paradigm assumes that the genotype of an insect-pathogenic fungus coevolves with a certain taxon of insect host that it infects. The insect-pathogenic fungus Beauveria bassiana shows a wide host range and is considered to be a facultative insect pathogen. In this study, a population genetics analysis of B. bassiana from forested and agricultural habitats as well as from the Canadian Arctic showed distinct genetic groups associated with the three different habitats. Within each group, recombining population structures and clonally reproducing lineages were observed. The B. bassiana isolates were also assessed for their abilities to grow at 8, 15, 25 and 37 degrees C and for their tolerances to UV exposure. The genetic groups from the Arctic and from the forested habitats grew at lower temperatures, while the genetic group from the agricultural habitat grew at 37 degrees C and was tolerant to UV exposure. There were no clear associations between the genetic group and the ability to infect coleopteran or lepidopteran insect larvae. There is increasing evidence that such studies represent a significant paradigm shift; habitat selection, not insect host selection, drives the population structure of deuteromycetous insect-pathogenic fungi. We suggest that adaptation to a certain habitat type is an important criterion for identifying insect-pathogenic fungal strains for use in insect biocontrol efforts.     

An improved method for Beauveria bassiana transformation using phosphinothricin acetlytransferase and green fluorescent protein fusion gene as a selectable and visible marker

An improved transformation method for the biocontrol agent, Beauveria bassiana, was developed. For convenience of transformation selection and detection, the coding regions of the genes for phosphinothricin acetyltransferase and green fluorescent protein were fused and an expression vector, pBFT, carrying this fusion was constructed. Under optimum conditions, over 60 transformants microg(-1) plasmid DNA were obtained. B. bassiana conidia frozen 1 month at -80 degrees C were fully competent for transformation. The method was significantly less laborious and more rapid than current methods for B. bassiana. The bar::egfp provides a selectable and visible marker which may expedite future genetic engineering of this fungus.     

应用球孢白僵菌菌条持续控制松褐天牛效果的分子评价

采用Ⅰ型内含子技术对应用球孢白僵菌无纺布菌条持续控制松褐天牛的防治效果进行了分子生物学评价.结果表明: 采用常规手段评价防治效果,两个处理区的防治效果分别为19.5%和10.8%; 而采用分子手段进行评价,防治效果分别为14.2%和11%.证明常规评价以死亡率或侵染率来计算微生物防治效果,难以排除当地微生物区系中的土著菌株所造成的侵染死亡,而采用分子生物学手段进行防治效果评价可从一定程度上消除土著菌株的干扰,较准确地反映人工放菌的防治效果.     

Diversity of Burkholderia isolates from woodland rhizosphere environments

AIMS: Determination of genetic diversity among UK Burkholderia cepacia isolates from various environmental niches, principally woodland tree rhizospheres and onions. METHODS AND RESULTS: Genus determination was made using polymerase chain reaction (PCR) amplification and fatty acid methyl ester profiling. Genetic diversity was investigated by repetitive sequence genetic PCR fingerprinting. Several onion isolates were similar to clinical isolates but others were diverse. Some environmental isolates were possibly synonymous with B. cepacia and B. gladioli but most from woodland rhizospheres were distinct and clustered together. The 16S rRNA genes of representatives from these clusters were PCR amplified, sequenced and phylogenetically compared with all known Burkholderia and related species. This revealed that the rhizospheric isolates had closest affinity with Burkholderia spp. with known bioremediative and biocontrol capabilities and were unrelated to taxa comprising plant or human pathogenic strains. CONCLUSIONS: All of the analyses investigated revealed that environmental and onion isolates of B. cepacia complex bacteria are genetically diverse but that woodland rhizospheric isolates are related to each other and unrelated to plant or human pathogenic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Woodland rhizospheric isolates of B. cepacia are potentially good candidates for use in bioremediation and biocontrol, as they appear distinct from plant or human pathogenic strains.     

球孢白僵菌种群在松林中的寄主转移及遗传多样性对松毛虫持续控制的影响

通过林间接种式放菌及其后一周年的野外调查,从林间采集到30种寄主昆虫及从土壤、落叶和气流中分离到119株球孢白僵菌．酯酶同工酶分析表明,它们属于32个不同酯酶型,呈现出丰富的遗传多样性．释放菌株所属的酯酶型02包括从11种昆虫上分离出的18个菌株,表明林间释放的菌株已成功地在不同寄主昆虫种群中宿存下来,并以常发的地方病状态存在于松毛虫及松灰象甲等12种昆虫种群之中;当林间目标寄主缺乏时,其它寄主可将食物链维系下去．其它酯酶型分别包括1～23个菌株．一周年内的寄主转移动态结果表明,球孢白僵菌在松林生态系统中不同寄主间可转移寄生．每个酯酶型中的菌株对松毛虫的毒力相差很大,表明球孢白僵菌在松林中的延续和扩散流行不是1条路线,每个酯酶型至少代表食物网上的1条支链．有些环节的寄主连接了不同的酯酶型,使松林中食物网变得十分复杂,另外。从土壤、枯枝落叶层、林冠层和空气中分离到的球孢白僵菌分属于不同的酯酶型,表明松林中还存在着复杂的腐生食物链,有利于松毛虫及其它害虫的持续控制。     

Molecular studies of co-formulated strains of the entomopathogenic fungus,Beauveria bassiana

A 28S rDNA intron was used as a molecular marker to distinguish between two single spore strains of Beauveria bassiana, Bb123 and Bb151. When co-formulated and assayed against larvae of Galleria mellonella these strains exhibited no synergistic increase in virulence, rather Bb123 usually dominated. This study shows that the success of any strain to infect Galleria is dependent on the dose and method of inoculation (injection versus immersion). The result of co-formulated strains grown on solid culture also showed that usually one strain dominated, i.e., strain displacement could happen both in vivo and in vitro. The speed by which one strain was displaced following successive sub-culturing on PDA partly depended on the ratio of Bb151 and Bb123. The co-formulated inoculum could widen the window over which parent strains would be active on different water activity media. Co-infection did result in heterokaryosis within the Galleria host. Molecular studies also showed that the heterokaryon was not stable and could revert back to the parent strain.     

Field-based evaluation of biopesticide impacts on native biodiversity: malagasy coleoptera and anti-locust entomopathogenic fungi

A community of 225 species of Coleoptera was used as a surrogate to evaluate nontarget effects of entomopathogenic fungi under development as biopesticides for use against the Malagasy migratory locust Locusta migratoria capito Saussure (Orthoptera: Acrididae). Evaluation of a standard chemical treatment of fenitrothion + esfenvalerate, two indigenous isolates of Metarhiziumflavoviride Gams & Roszsypol (SP3 and SP9), and an indigenous isolate of Beauveria bassiana (Balsamo) Vuillemin (SP16) against an untreated control in a replicated field trial in southern Madagascar showed that one of the isolates of M. flavoviride (SP3) and fenitrothion + esfenvalerate had distinct effects on nontarget beetle communities that were similar to each other. The other two isolates had no detectable effects compared with the untreated control. Based on an evaluation of the species affected, the similar effects of SP3 and the chemical pesticide are hypothesized to be the result of a perturbation of predator-prey relationships, with a distinct tendency to be manifested via predators. The data indicate that use of SP9 and SP16 would have minimal detrimental effects on the biodiversity of nontarget beetles, but that SP3 needs further testing.     

Phenotypic and genetic characterization of Paecilomyces lilacinus strains with biocontrol activity against root-knot nematodes

Efficient selection of fungi for biological control of nematodes requires a series of screening assays. Assessment of genetic diversity in the candidate species maximizes the variety of the isolates tested and permits the assignment of a particular genotype with high nematophagous potential using a rapid novel assay. Molecular analyses also facilitate separation between isolates, allowing the identification of proprietary strains and trace biocontrol strains in the environment. The resistance of propagules to UV radiation is an important factor in the survival of a biocontrol agent. We have analyzed 15 strains of the nematophagous fungus Paecilomyces lilacinus using these principles. Arbitrarily primed DNA and allozyme assays were applied to place the isolates into genetic clusters, and demonstrated that some genetically related P. lilacinus strains exhibit widespread geographic distributions. When exposed to UV radiation, some weakly nematophagous strains were generally more susceptible than effective isolates. A microtitre tray-based assay used to screen the pathogenic activity of each isolate to Meloidogyne javanica egg masses revealed that the nematophagous ability varied between 37%-100%. However, there was no clear relationship between nematophagous ability and genetic clusters. Molecular characterizations revealed sufficient diversity to allow tracking of strains released into the environment.     

两广地区家蚕白僵菌的SSRs遗传多样性

为查明广东和广西地区家蚕Bombyx mori病原白僵菌的来源及其菌株间的相互关系, 本研究利用了微卫星标记（simple sequence repeats, SSRs）技术, 分别对采自广东、 广西蚕区的白僵菌菌株居群之间和居群之内的遗传多态性进行了研究。结果发现, 两广白僵菌居群之间的基因分化系数（Gst）是0.0590, 多态位点百分率（PPL）为97.73%, Nei氏基因多样性指数（H）为0.1896, Shannon氏信息多样性指数（I）为0.3165, 表明两广家蚕来源的白僵菌居群间的遗传分化较小; 两个居群内部的遗传多态性研究结果分别是广东白僵菌居群的PPL=68.18%, H=0.1910, I=0.3044, 而广西白僵菌居群的PPL=65.91%, H=0.1713, I=0.1791, 表明广东白僵菌居群的遗传多样性水平较高, 广西白僵菌居群遗传多样性水平相对较低。最后, 利用Nei氏遗传距离进行了两广地区白僵菌菌株间地理来源关系的聚类分析, 结果表明实验室保存菌株单独聚为类群Ⅰ, 而不同采集地的菌株聚为类群Ⅱ。结果反映了生产来源的白僵菌菌株存在遗传多态性和基因分化现象, 暗示了家蚕白僵病病原来源的复杂性, 还说明应用SSRs技术进行家蚕白僵病病原的溯源是一条可行的途径。     

Beauveria bassiana as a pathogen of the Mexican fruit fly (Diptera: Tephritidae) under laboratory conditions

Bioassays were carried out under controlled conditions (27 +/- 2 degrees C, 80 +/- 5% RH, and a photoperiod of 12:12 [L:D] h) to evaluate the effect of eight strains of the entomopathogenic fungus Beauveria bassiana upon larvae, pupae, and adult females of the Mexican fruit fly, Anastrepha ludens (Loew). Mortality of the immature stages was low, 2-8% in larvae and 0% in pupae. However, very high levels of mortality were obtained for adult flies, with values of 100, 98, and 98% for the strains Bb16, Bb24, and Bb26, respectively. LC50 values for these three strains ranged from 3.12 x 10(6) to 9.07 x 10(6) conidia/ml. Lethal time 50 (LT50) was 2.8, 3.7, and 4.2 d for Bb16, Bb26, and Bb24 strains, respectively, with an average LT50 of 4.4 d across all strains. The fungal mycelium emerged through the soft parts of the exoskeleton, such as the wing bases, mouth, intersegmental regions of the legs, and membranous regions of the abdomen, coxae, and neck. Maximum percentage sporulation ranged from 66.4 to 74.7% for the three most virulent strains.     

Molecular investigation on strain genetic relatedness and population structure of Beauveria bassiana

Triplicate molecular methods, i.e. polymerase chain reaction-restriction fragment length polymorphism of the pr1 gene, microsatellite markers and 28S rDNA haplotyping by detecting the presence or absence of group I introns, were used for population study of the entomopathogenic fungus, Beauveria bassiana. The findings showed that the average genetic diversity index of geographical populations was significantly smaller than that of populations derived from insect host orders, indicating that the genetic relatedness of B. bassiana strains was highly associated with geographical locality rather than insect host species. The reproductive style of all the B. bassiana populations was found to be non-clonal. Population structure analysis revealed that the average divergent coefficient among populations of B. bassiana was far below 1 (0.1112), which indicated that there was no significant genetic differentiation between populations, and that the overall genetic diversity mainly resulted from the genetic variations within geographical populations. Statistically, genetic distances between populations were positively correlated with geographical distances, suggesting that geographical separation poses an obstacle to the possibility and frequency of genetic exchanges between populations. On the other hand, gene flow was indirectly established to occur between B. bassiana populations.     

Beauveria bassiana -A Potential Mycopesticide for the Efficient Control of Coffee Berry Borer, Hypothenemus hampei (Ferrari) in India

The Coffee Berry Borer (CBB), Hypothenemus hampei (Ferrari) (Coleoptera: Scolytidae) has been a serious insect pest of coffee cultivars C. robusta and C. catimor in India since 1991, causing 40-80% coffee bean loss. To combat this important pest, an indigenous entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin was isolated from dead and moribund coffee berry borers from the wild. The fungus was cultured on yeast extractpeptone supplemented liquid medium. The mycelial mat was harvested from 12-day old cultures and lyophilized. A suspension of the most virulent isolate (Bb2) was prepared in sterile water and used as a mycopesticide. The laboratory studies were conducted on coffee berry borers by applying conidial suspensions at a dosage rate of 1 ×10 6 conidia ml -1 . Pest mortality with the Bb2 isolate increased from 69.3% to 95.3% with an increase in relative humidity (RH) at 25 ±2°C. Field experiments were conducted in a coffee plantation area in the Kodagu district of Karnataka, and the results showed that, under favourable environmental conditions (27- 29°C; 82-91% RH; 10-15 inches rainfall per year), the fungus required only eight days to colonize and kill the target pest. A maximum of 75.6% insect mortality was recorded 24 days after spraying. Large-scale field trials conducted in five plantation plots between September 1995 and September 1998 showed significant insect mortality both in C. robusta and C. catimor cultivars of coffee. The potential use of this indigenous fungal strain of B. bassiana as a mycopesticide for management of CBB in India is discussed.     

外来树种对本地林业虫害的诱发作用

白蜡窄吉丁、萧氏松茎象、光肩星天牛、黄斑星天牛、云斑天牛和桑天牛等林业害虫均为我国本地林业生态系统中的昆虫种类,在北美白蜡树、北美松树和北美黑杨派杨树等外来树种引入和大量栽培之前,一直没有形成严重的危害,以至于白蜡窄吉丁和萧氏松茎象都没有引起人们的关注。这些重要林业害虫在我国的发生都是伴随着敏感外来树种的引进和大量不合理的种植而逐渐严重起来的。本文对外来树种的引进与本地林业虫害的发生之间的关系进行了分析,提出了外来树种的不合理引进可能诱发本地林业新虫害的观点,并对其发生机理以及相应的研究和治理对策进行了讨论。     

Growth-inhibiting effects of concentrations of fusaric acid on the growth of Bacillus mojavensis and other biocontrol Bacillus species

AIMS: To determine the effects of concentrations of fusaric acid on the growth of several strains of the biocontrol bacterial endophyte Bacillus mojavensis and other species within the Bacillus subtilis group, as well as the genetic relationships within this small group of Gram-positive bacteria, and their antagonisms to Fusarium verticillioides, which produce fusaric acid. METHODS AND RESULTS: The growth of 50 Bacillus strains and species were tested at two concentrations of fusaric acid determined in maize infected by an isolate of F. verticillioides. Molecular characterizations of the strains and species of bacteria were determined with an automated ribotyper. The growth of bacteria measured under both concentrations with an automated turbidometer, Bioscreen, indicated that fusaric acid was toxic to most strains of the bacterial endophyte B. mojavensis. However, the effects of these two concentrations on other Bacillus species varied in that fusaric acid was either bacteriocidal or bacteriostatic to most species. CONCLUSIONS: These data indicate that the concentrations of fusaric acid are inhibitory to the growth of most Bacillus species, some of which are used as biocontrol agents. This suggests that the endophytic and saprophytic states of F. verticillioides and other Fusarium species cannot be controlled by fusaric-acid-sensitive Bacillus species. SIGNIFICANCE AND IMPACT OF STUDY: Mycotoxic Fusarium species, such as F. verticillioides, are competitive because all produce fusaric acid, which is inhibitory to biocontrol bacteria, and mutants tolerant to fusaric acid must be developed in order to be effective on biocontrol bacteria.     

Safety of Microorganisms Intended for Pest and Plant Disease Control: A Framework for Scientific Evaluation

Microorganisms are enormous but largely untapped natural resources for biological control of pests and diseases. There are two primary reasons for their underployment for pest or disease control: (1) the technical difficulties of using microorganisms for biological control, owing to a lack of fundamental information on them and their ecology, and (2) the costs of product development and regulatory approvals required for each strain, formulation, and use. Agriculture and forestry benefit greatly from the resident communities of microorganisms responsible for naturally occurring biological control of pest species, but additional benefits are achieved by introducing/applying them when or where needed. This can be done as (1) an inoculative release, (2) an augmentative application, or (3) an inundative application. Because of their specificity, different microbial biocontrol agents typically are needed to control different pests or the same pest in different environments. Four potential adverse effects are identified as safety issues (hazards) associated with the use of microorganisms for the biological control of plant pests and diseases. These are: (1) displacement of nontarget microorganisms, (2) allergenicity to humans and other animals, (3) toxigenicity to nontarget organisms, and (4) pathogenicity to nontarget organisms. Except for allergenicity, these are the same attributes that contribute to the efficacy of microbial biocontrol agents toward the target pest species. The probability of occurrence of a particular adverse nontarget effect of a microbial biocontrol agent may be a function of geographic origin or a specific trait genetically added or modified, but the safety issues are the still the same, including whether the microorganism intended for pest or disease control is indigenous, nonindigenous (imported and released), or genetically modified by traditional or recombinant DNA (rDNA) technology. Likewise, the probability of occurrence of a particular adverse nontarget effect may vary with method of application, e.g., whether as an aerosol, soil treatment, baits, or seed treatment, and may increase with increased scale of use, but the safety issues are still the same, including whether the microorganism is used for an inoculative release or augmentative or inundative application. Existing practices for managing microorganisms in the environment (e.g., plant pathogens,Rhizobium,plant inoculants) provide experience and options for managing the risks of microorganisms applied for pest and disease control. Moreover, experience to date indicates that any adverse nontarget effects, should they occur, are likely to be short-term or transitory effects that can, if significant, be eliminated by terminating use of the microbial biocontrol agent. In contrast, production agriculture as currently practiced, such as the use of tillage and crop rotations, has significant and long-term effects on nontarget organisms, including the intentional and unintentional displacement of microorganisms. Even the decision to leave plant pests and diseases unmanaged could have significant long-term environmental effects on nontarget organisms. Potential safety issues associated with the use of microbial biocontrol must therefore be properly identified and compared with the impact of other options for managing the pest or leaving the pest unmanaged. This paper provides a scientific framework for this process.