感染松墨天牛的金龟子绿僵菌菌株的初步筛选

松墨天牛是松材线虫病的主要媒介昆虫,本文对分离自松墨天牛的6株金龟子绿僵菌,以及从光肩星天牛、大蜡螟、伊藤厚丝角叶蜂和土蝽上分离的各1个菌株,共10个菌株的产孢情况、孢子萌发率进行研究;在此基础上选出金龟子绿僵菌1291、1349和2049三个菌株及球孢白僵菌F-263菌株,采用成虫跗节接种法和幼虫浸渍法进行室内松墨天牛及大蜡螟的毒力测定比较。结果表明,供试绿僵菌菌株对松墨天牛幼虫在接种15天后的感染僵虫率为76.9％～93.1％(1×10⁷孢子/mL);成虫在接种20天后的僵虫率亦达57.9％～75.0％(6.5×10⁵～3.4×10⁶孢子/成虫);2049菌株表现尤其突出;对应的球孢白僵菌F-263对幼虫和成虫的僵虫率分别是96.3％(1×10⁷孢子/mL)和55％(9.7×10⁵孢子/成虫)。但这3个金龟子绿僵菌菌株对大蜡螟的毒力较低,存在较明显的寄主专化性。这3个菌株今后在防治松墨天牛方面具有较大开发应用价值。     

金龟子绿僵菌MaYTTR-04菌株对松墨天牛成虫的致病力

松墨天牛Monochamus alternatus Hope是重大森林植物检疫性病害——松材线虫病的主要媒介昆虫。采用跗节接种法测定金龟子绿僵菌Metarhizium anisopliae MaYTTR-04菌株对松墨天牛成虫的致病力。结果表明： 在恒温条件下,松墨天牛第6天开始死亡,第18天～21天为死亡高峰期,每头成虫接种孢子量为2.3×10⁶±0.2×10⁶时成虫的死亡率第18天达85%,第21天达95%; 时间效应指标值LT₅₀为14.7天。室内自然变温条件下,松墨天牛第3天出现死亡,第15天～21天为死亡高峰期,每头成虫接种孢子量为2.3×10.6±0.2×10⁶时的死亡率在第15天达85%,第21天达100%;时间效应指标值LT₅₀为12.9天。林间套笼21天后,菌液处理的成虫平均死亡率为60%,僵虫率为48.9%;而通过无纺布菌条的成虫死亡率为86.7%,僵虫率为75.6%。表明MaYTTR-04菌株对松墨天牛成虫有强的致病力,且在较高变温环境温度下致病力更强。结果说明该菌株可作为生产性菌株应用于林间防治松墨天牛。     

Colonization of Metarhizium anisopliae on the surface of pine tree logs: A promising biocontrol strategy for the Japanese pine sawyer,Monochamus alternatus

We investigated the colonization potential of five Metarhizium anisopliae isolates on pine tree surfaces under laboratory conditions, determined the influence of the pine bark extract on fungal growth and evaluated the insecticidal activity following colonization on the Japanese pine sawyer. Finally, the effect of colonization on adults pine sawyer was evaluated using the top three performing isolates (JEF-197, JEF-271 and JEF-279) under laboratory and field conditions. As a result, isolate JEF-197 showed the highest conidial production on the pine surfaces, and five isolates, including JEF-197, showed higher hyphal growth on autoclaved pine bark extract agar, compared to a water agar. Pine bark treated with the isolates showed 40–70 % mortality of adults pine sawyer. Under mimicked overwintering conditions, in the JEF-197 treatment group, 40 % of the inserted larvae became adults and all were dead after 59 d. In a field test, colonized isolate JEF-197 also showed 37 % insecticidal activity against emerged adults from the pine logs as overwintering sites. This work suggests that M. anisopliae isolate JEF-197 possibly colonized the pine surface and application of a conidial suspension on the pine logs as overwintering sites could be an effective strategy to control the pine sawyer.     

Effect of cultival practices and soil treatments on incidence of wilt disease of pigeon pea

Summary The incidence of wilt disease of pigeon pea (Cajanus cajan (L.) Millsp.) caused byFusarium udum under soil treatments with various substances was studied under field conditions. The disease incidence was highly suppressed under mixed cropping withCrotalaria medicaginea. Phygon XL was found to be the most effective fungicide to reduce the incidence of the disease. The incidence of wilt disease also decreased in case of soil amended with the leaves ofC. medicaginea. Wilt incidence increased in the soil amended with the roots of pigeon pea.     

Pine wilt disease in Yunnan,China: Evidence of non-local pine sawyer Monochamus alternatus (Coleoptera: Cerambycidae) populations revealed by mitochondrial DNA

Abstract Monochamus alternatus (Hope) specimens were collected from nine geographical populations in China, where the pinewood nematode Bursaphelenchus xylophilus (Steiner et Buhrer) was present. There were seven populations in southwestern China in Yunnan Province (Ruili, Wanding, Lianghe, Pu’er, Huaning, Stone Forest and Yongsheng), one in central China in Hubei Province (Wuhan), and one in eastern China in Zhejiang Province (Hangzhou). Twenty-two polymorphic sites were recognized and 18 haplotypes were established by analyzing a 565 bp gene fragment of mitochondrial cytochrome oxidase subunit II (CO II). Kimura two-parameter distances demonstrated that M. alternatus populations in Ruili, Wanding and Lianghe (in southwestern Yunnan) differed from the other four Yunnan populations but were similar to the Zhejiang population. No close relationship was found between the M. alternatus populations in Yunnan and Hubei. Phylogenetic reconstruction established a neighbor-joining (NJ) tree, which divided haplotypes of southwestern Yunnan and the rest of Yunnan into different clades with considerable bootstrapping values. Analysis of molecular variance and spatial analysis of molecular variance also suggested significant genetic differentiation between M. alternatus populations in southwestern Yunnan and the rest of Yunnan. Our research suggests that non-local populations of M. alternates, possibly from eastern China, have become established in southwestern Yunnan.     

Impact of fungicides on Metarhizium anisopliae in the rhizosphere, bulk soil and in vitro

The entomopathogenic fungus Metarhizium anisopliae (Metchnikoff) Sorokin (Hypocreales: Clavicipitaceae) is registered in the United States and The Netherlands for black vine weevil, Otiorhynchus sulcatus (Coleoptera: Curculionidae) control in container-grown ornamentals. These studies were conducted to determine the compatibility of M. anisopliae (F52) with a wide range of fungicides commonly applied to container-grown ornamentals for the management of soil-borne plant pathogens. The impact of fungicides on spore germination and mycelial growth were determined in vitro. In addition, M. anisopliae persistence in bulk and rhizosphere soil was determined 30 days following dual application of each fungicide at 7–28 days intervals as prescribed. A number of fungicides (thiophanate-methyl, dimethomorph, captan, triflumizole, triflozystrobin, pyraclostrobin, azoxystrobin) inhibited spore germination in vitro. A larger number of fungicides (fosetyl-AI, thiophanate-methyl, dimethomorph, captan, quintozene, triflumizole, fludioxanil, triflozystrobin, pyraclostrobin, fludiox-mefanox, iprodione, azoxystrobin, phosphorus acid/K-salts) inhibited mycelial growth in vitro. Only three fungicides (etridiazole, propamocard and mafanoxam) had no significant impact in vitro on spore germination or mycelial growth. While a number of fungicides had a detrimental impact in vitro, there was no impact on M. anisopliae populations in bulk soil following dual application of any fungicide. However, the fungicides captan and triflumizolet, which have a short reapplication interval, had a detrimental impact on M. anisopliae populations in the rhizosphere. As researchers develop rhizosphere competence as an alternative management strategy for black vine weevil, the fungicides captan and triflumizole should be avoided.     

Population density ofMonochamus alternatus adults (Coleoptera: Cerambycidae) and incidence of pine wilt disease caused byBursaphelenchus xylophilus (Nematoda: Aphelenchoididae)

Summary The seasonal occurrence ofMonochamus alternatus and newly weakened trees were investigated in aPinus thunbergii stand for 4 years. Adult beetles were present between June and September with a peak in their population occurring in early July followed by a decline then a period of about one month being in a steady number. The average number ofBursaphelenchus xylophilus (Nematoda), which is the causal agent of pine wilt disease, within beetles decreased as the season advanced. Pine trees newly weakened byB. xylophilus appeared between June and October, especially from August to October. The proportion of weakened or killed trees was directly proportional to the average beetle density per tree from June to August.     

Relationship between soil properties and incidence of pine wilt disease at stand level

This study was conducted to evaluate and compare soil properties between pine stands either damaged or undamaged by pine wilt disease in Jinju and Sacheon Cities, which are areas in Korea severely affected by the disease. Soil physical and chemical properties were generally similar between damaged and undamaged pine stands. There was no significant difference in soil factors related to soil nutrient fertility between damaged and undamaged pine stands, although the incidence of pine wilt disease was affected by soil clay content, which was significantly (P < 0.05) higher in the damaged (24.8%) than in the undamaged (20.1%) pine stands. However, this relationship should be treated with caution because small scale topographical variation may simply reflect the incidence of pine wilt disease. The results suggest that incidence of pine wilt disease was not influenced by soil nutrient properties at a small stand scale.     

Evaluation of Metarhizium anisopliae (Metsch) Sorok. to target larvae and adults of Capnodis tenebrionis (L.) (Coleoptera: Buprestidae) in soil and fiber band applications

The aim of this work has been to evaluate in the laboratory the potential of entomopathogenic fungi against adults and larvae of Capnodis tenebrionis (L.) (Coleoptera: Buprestidae) through fiber band application and a potted plant bioassay with soil application, respectively. Our previous findings revealed that Metarhizium anisopliae EAMa 01/58-Su isolate was the most virulent against neonate larvae of the buprestid. In the present work, M. anisopliae EAMa 01/58-Su isolate has been also shown to be highly virulent against adult beetles by immersion in a conidial suspension; thus it was selected to accomplish our objectives. When adult beetles were stimulated to climb 100 x 200 mm non-woven commercial fiber bands impregnated with conidia of M. anisopliae EAMa 01/58-Su isolate, total mortality rates varied from 85.7% to 100.0%; whereas no significant correlation was detected between the time needed to cross the band (mean value 648.7+/-22.4s) and the time of death, with mean average survival time ranging between 10.3 and 16.0 days, compared to 28 days of the controls. Potted seedlings (5-6 months old) of cherry plum (Prunus myrobalana Lois.), a commonly used apricot rootstock, were used to study the efficacy of soil treatment with M. anisopliae EAMa 01/58-Su isolate against neonate C. tenebrionis larvae. The soil inoculation with M. anisopliae EAMa 01/58-Su isolate had a significant effect on the mean number of dead larvae recovered from the roots, with mean mortality ranging from 83.3% to 91.6%; whereas no significant differences were detected between the three fungal doses. In all cases, dead larvae found within roots exhibited external signs of fungal growth. Hence, it may be possible to use M. anisopliae EAMa 01/58-Su isolate in a biocontrol strategy targeting both adults and larvae of C. tenebrionis.     

树木生长势与松墨天牛种群密度及松材线虫发病程度关系

对不同生长势林区内的松墨天牛种群密度和松材线虫发病程度的研究表明,树木生长势（x）分别与松墨天牛种群密度（y）、松材线虫发病程度（z）呈负相关,其线性回归方程分别为：y=1793.771-16404.47x;z=31.80989-241.9274x,相关系数分别为r=-0.8319和r=-0.8770。而松墨天牛种群密度（y）与松材线虫发病程度（z）呈正相关,其线性回归方程为：y＝－407.0611＋70.51478z;相关系数为r=0.9864。     

Laboratory and field studies for the control of Chagas disease vectors using the fungus Metarhizium anisopliae

Chagas disease is one of the most important insect-vectored diseases in Brazil. The entomopathogenic fungus Metarhizium anisopliae was evaluated against nymphs and adults of Panstrongylus megistus, Triatoma infestans, and T. sordida. Pathogenicity tests at saturated humidity demonstrated high susceptibility to fungal infection. The shortest estimates of 50% lethal time (LT₅₀) for P. megistus varied from 4.6 (isolate E9) to 4.8 days (genetically modified strain 157p). For T. infestans, the shortest LT₅₀ was 6.3 (E9) and 7.3 days (157p). For T. sordida, the shortest LT₅₀ was 8.0 days (157p). The lethal concentration sufficient to kill 50% of T. infestans (LC₅₀) was 1.9 × 10⁷ conidia/ml for strain 157p. In three chicken coops that were sprayed with M. anisopliae, nymphs especially were well controlled, with a great population reduction of 38.5% after 17 days. Therefore M. anisopliae performed well, controlling Triatominae in both laboratory and field studies.     

Effect of soil temperature and moisture on survival and infectivity of Metarhizium anisopliae to four tephritid fruit fly puparia

The infectivity of 4 isolates of Metarhizium anisopliae to puparia of Ceratitis capitata treated as late third-instar larvae in unsterilized soil was investigated in the laboratory under controlled temperature and moisture. At 20-30 degrees C, mortality in puparia was highest at water potential of -0.1 and -0.01 mega Pascal (MPa) and lowest at water potential of -0.0055 and -0.0035 MPa in all the isolates. In wetter soil however, isolates ICIPE 20 and 60 caused significantly higher mortality than ICIPE 18 and 69. The survival of conidia in drier soil (-0.1 MPa) was not adversely affected at all temperatures. However, in wet soil (-0.0035 MPa) there was drastic reduction in colony counts in ICIPE 18 and 69 at 25 and 30 degrees C but conidial density in ICIPE 20 and 60 remained at the initial level at 14 days after inoculation at all temperatures. When ICIPE 20 was evaluated against three other fruit fly species (Ceratitis cosyra, Ceratitis rosa, and Ceratitis fasciventris), significant reduction in adult emergence and higher pupal mortality occurred in C. cosyra and C. fasciventris than in C. rosa at a combination of 15 and 20 degrees C and -0.1 and -0.0035 MPa. However, at higher temperature and the same moisture level, the isolates were equally pathogenic across the 3 species. It is probable that in addition to pathogen cycling and multiplication from dead infected insects in the soil, a balance between microbial degradation and replenishment of inoculum of virulent isolates occur through fluctuations in, and intricate interactions between temperature and moisture levels. This study is indicative of the potential of using isolate ICIPE 20 for soil inoculation against pupariating third-instar larva of fruit flies, thus providing a novel alternative to chemical soil application.     

Susceptibility of Phyllophaga polyphylla and Anomala cincta larvae to Beauveria bassiana and Metarhizium anisopliae isolates, and the interaction with soil properties

The white grub species Phyllophaga polyphylla and Anomala cincta (Coleoptera: Melolonthidae) are economically important species that affect many crops in Mexico. A series of experiments to study the pathogenic interaction between isolates of Beauveria bassiana and Metarhizium anisopliae and these two insect species were undertaken. First, the susceptibility of third instar P. polyphylla larvae to each of seven isolates representing both species of fungus was evaluated by dipping the insects in 1?×?10⁸ conidia?ml^?1 suspensions. A second study examined the differences in the susceptibility of P. polyphylla and A. cincta larvae to two selected isolates for each of the fungal species. Finally, the susceptibility of A. cincta larvae to one M. anisopliae isolate when incubated in soil collected from four different sites was assessed. No significant differences in proportion of infection of P. polyphylla larvae were observed amongst the fungal isolates tested and mortality due to fungal infection was never greater than 20% after 36?days incubation. Anomala cincta larvae were more susceptible than P. polyphylla larvae, with greater than 90% infection when inoculated with isolates of M. anisopliae whereas mortalities of only 20% where achieved against P. polyphylla larvae. The soil type in which A. cincta were incubated following inoculation with M. anisopliae affected their susceptibility to infection. The results demonstrated that there is a complex interaction amongst entomopathogenic fungi, white grub larvae and soil properties, and points to the need of further investigation of this system in order to optimize the efficacy of entomopathogenic fungi against these insect species.     

Efficacy of soil application of Metarhizium anisopliae and the use of GF-120 spinosad bait spray for suppression of Bactrocera invadens (Diptera: Tephritidae) in mango orchards

Field trials were conducted for two seasons in mango orchards at Nthagaiya, Kenya, to evaluate the efficacy of soil inoculation with Metarhizium anisopliae (Hypocreales: Clavicipitaceae) and GF-120 spinosad bait sprays, applied either alone or in combination, for suppression of the invasive fruit fly, Bactrocera invadens (Diptera: Tephritidae). During the 2006/2007 season, average post-treatment samples showed that B. invadens catches from the control orchards were four times higher than the number of flies captured in the plots receiving M. anisopliae+GF-120. Fruit infestation was 16, 45, 30 and 60% in the M. anisopliae+GF-120, M. anisopliae alone, GF-120 alone and control orchards, respectively. In the 2007/2008 season, average B. invadens post-treatment samples in the control orchards were seven times higher than the treatment with M. anisopliae+GF-120; and fruit infestation was 11, 38, 28, and 52% in the orchards assigned to M. anisopliae+GF-120, M. anisopliae alone, GF-120 alone and control treatments, respectively. Enumeration of conidial densities from soil samples on agar plates showed initial densities of 1.1–2.1×10⁵ colony forming units (cfu)/g of dry weight of soil but decreased to 1.0–1.4×10³ cfu/g at the end of the experimental period. Exposure of laboratory-reared pupariating larvae to soil samples taken from fungus-treated fields showed significant reduction in B. invadens adult emergence (25–36%) compared with the control (80–82%). Our results suggest that the combined use of soil application of M. anisopliae and GF-120 spinosad bait spray is an effective IPM strategy for field suppression of B. invadens on mango.     

Cold tolerance and cold hardening strategy of the Japanese pine sawyer Monochamus alternatus (Coleoptera: Cerambycidae)

The Japanese pine sawyer, Monochamus alternatus , is an important pine forest pest and vector transmitting the pine wilt nematode that causes pine wilt disease. Low temperatures in autumn, winter and spring often differentially affect mortality of M. alternatus larvae. In this paper, we mainly compared the differences of mortality and cold hardening of larvae from different seasons, based on supercooling point (SCP) and cumulative probability of individuals freezing (CPIF). The cold hardening of the larvae from autumn, winter and spring seasons were largely different. Correlations between mortality and CPIF of autumn and spring larvae were highest on day 1/4, and gradually decreased with prolonged exposure duration. This beetle's death mainly resulted from freezing in short exposure duration. However, the correlation between mortality and CPIF of winter larvae increased gradually with the prolonged exposure duration. Death did not mainly result from freezing in long exposure duration. Autumn larvae are more susceptible and adaptable than winter and spring larvae. Winter larvae have a slight freeze-tolerance trend. Our research showed that M. alternatus came into complex cold-hardening strategies under natural selection. Freeze avoidance is the primary strategy; with prolonged exposure duration to above SCP or < 0 °C, chill tolerance is more important; this is followed by freeze tolerance during harsh winters.     

Morphology and distribution of antennal sensilla in the pine wood nematode insect vectors Monochamus alternatus Hope and Monochamus saltuarius Gebler (Coleoptera: Cerambycidae)

As insect vectors of pine wood nematodes, Monochamus alternatus Hope and Monochamus saltuarius Gebler have different levels of attractiveness to semiochemicals. Although active use of chemical communication in both species is well known, there was no report on the differences in morphology and distribution of antennal sensilla between the two Monochamus species. The present study was carried out to explore the morphology, distribution, and ultrastructure of antennal sensilla of the two species using scanning and transmission electron microscopy. The overall shapes of the antennae were not different between M. alternatus and M. saltuarius. However, both M. alternatus and M. saltuarius exhibited sexual dimorphism overall. The antennae of each species were composed of the scape, pedicel, and nine flagellomeres, with four morphological types of sensilla: trichodea, basiconica, chaetica, and coeloconica. The four types occurred in varying numbers and distributions. The sensilla chaetica type was the most abundant in both species. Three types of sensilla (trichodea, basiconica, and chaetica) could be subdivided according to the two Monochamus species; sensilla trichodea could be divided into two subtypes, and sensilla basiconica and sensilla chaetica into three. All sensilla basiconica subtypes were multiporous, indicating that their primary function was olfactory. Sensilla trichodea subtype 2 was uniporous, indicating that the function of these sensilla might be gustatory. The morphological information obtained in our study provides a basis for future investigation of the sensory physiological function of each type of sensilla in the two species.     

Effects of pine wilt disease invasion on soil properties and Masson pine forest communities in the Three Gorges reservoir region,China

Pine wilt disease (PWD) has caused significant Masson pine mortality in the Three Gorges reservoir region in central China. In this study, five uniform Masson pine stand types infected by PWD were selected and surveyed on slopes and aspects with similar environmental conditions. In sites that had been infected, soil bulk density was reduced, and the difference among the groups was statistically significant (P < 0.05) at the 0–10 cm and 10–20 cm soil layers, but not at 20–40 cm. Other soil water‐related physical properties, excluding noncapillary porosity, significantly differed among the groups in all soil layers. Additionally, the values of available phosphorus, sodium, potassium, calcium, and magnesium were higher in the invaded stands, but the total nitrogen and organic matter contents were lower. Masson pine does not become reestablished following PWD‐induced mortality but is instead replaced by broad‐leaved tree species. Among the 19 examined environmental variables, five were found to be significantly related with the ordination of plant community structure: Masson pine stumps (MPS), K⁺, capillary water holding capacity (CWHC), capillary porosity (CP), and soil water content (SWC). Among these factors, the plant community structure was principally related to MPS and K⁺. The findings of this study show that the outbreak of PWD has impacted Masson pine forest soil properties and altered forest community composition. The disease is negatively related with the presence of Masson pine and positively associated with that of broad‐leaved tree species.     

Effects of conidial densities and spray volume of Metarhizium anisopliae and Beauveria bassiana fungal suspensions on conidial viability,droplet size and deposition coverage in bioassay using a novel bioassay spray system

A newly developed spray tower was used to characterise droplet distribution and coverage of conidial suspensions of Metarhizium anisopliae ATCC 62176 and Beauveria bassiana NI8 with different spray volumes. ATCC 62176 and NI8 had different spray models which could be caused by the surface physicochemical characteristics of the strains and conidia.     

Persistence of Conidia of Metarhizium anisopliae in Sugarcane Fields: Effect of isolate and formulation on persistence over 3.5 years

Metarhizium anisopliae is being used in Australia as a biopesticide for control of sugarcane whitegrubs in soil. The field persistence in sugarcane soil of two isolates of M. anisopliae each in four formulations was tested by mixing the formulation with soil which was then placed in PVC rings and buried in sugarcane fields. The two isolates used were FI-1045, M. anisopliae var. anisopliae, the active ingredient in BioCane™ currently used for greyback canegrub control, Dermolepida albohirtum (Coleoptera: Scarabaeidae: Melolonthinae), and FI-147, M. anisopliae var. lepidiotum, being tested as a biopesticide for Lepidiota spp. (Coleoptera: Scarabaeidae: Melolonthinae) and other species of canegrub. The four formulations were rice granules (as in BioCane™), a wettable powder derived from conidia screened from the rice granules, conidia off rice suspended in water and conidia produced on agar plates, dried, and then mixed with water for adding to soil. FI-1045 was tested at three different sites in north Queensland with a range of soil types and climatic conditions while FI-147 was tested at three similarly diverse sites in southern Queensland. The PVC rings were destructively sampled every 6 months for 3.5 years and the number of viable conidia remaining determined by plating onto a selective medium. The exponential decay was determined. Monthly decay rates ranged from 0.0309 to 0.0835 (mean 0.0512). A small proportion of conidia survived the 3.5 years at all sites and all formulations. Overall, isolate FI-147 persisted better than FI-1045, but was used at the more Southerly sites. Rainfall and soil type had negligible effects on persistence. The agar-produced FI-147 conidia declined most slowly, while the two rice-produced but water-formulated conidia gave similar results. Isolate FI-1045 survived best as the BioCane™ formulation and this rice granule formulation was almost as persistent as the agar conidia with FI-147. A small proportion of conidia, in some formulations and at some sites, were recovered from immediately below the rings. This movement was thought to be due to activity of earthworms or mites. The results suggest that 3 years would be the maximum period for a BioCane™ formulation to provide some level of infection in the target pest unless augmented by conidia from infected grubs. The effectiveness of these new conidia may be reduced due to their highly aggregated distribution.