Virulence and mycotoxic effects of Metarhizium anisopliae on Mahogany shoot borer,Hypsipyla robusta(Lepidoptera:Pyralidae)

Developing appropriate control measures for the Mahogany shoot borer, Hypsipyla robusta Moore has become increasingly important due to the severe damaging effect of the pest on the establishment of the saplings of Swietenia mahagoni Jacq （Sapindales： Meliaceae）. Existing management methods are largely limited to silvicultural practices and spraying of chemical insecticides. To identify a potential fungal biocontrol agent, we compared the virulence of six native and two standard ARSEF isolates of Metarhizium anisopliae Metsch. against this pest. The average survival time and conidial yield of IWST-Ma7 was higher （6.2 to 7.3 days and 4.9 to 4.7 x 105 conidia/ml） than the standards. Sig- nificant difference in sporulation on the cadavers between isolates, doses and incubation periods were substantiated for the selection of potential strain. The mycotoxic effects of crude soluble protein extract when in- corporated in the artificial diet, the ARSEF 2596 and ARSEF 3603 showed LDs0 value of 3.7% and 5.6%. However, IWST-Ma7 was highly lethal with significant lowest LDs0 value of 2.6%. The enzyme activity of IWST-Ma7 was highest for chitinase, CDA, protease and lipase viz., 1.90 U/mg, 1.80 U/mg, 0.98 U/mg and 0.80 U/mg respectively. However the enzyme activity of chitinase and Chitin deacetylase assay for all the isolates was significantly higher than protease and lipase activity. The ITS regions （5.8S rDNA and 28S rDNA） of seven isolates of M. anisopliae were amplified using the ITS1 and ITS4 primers which was a unique fragment of approximately 550 bp. Based on ITS regions, phy- logenetic tree have been constructed and the isolates have been grouped in to 5 clades. The virulence and mycotoxic effects of different isolates could rationally be used to employ them for the management of the mahogany borer.

Scanning Electron Microscopic Observation of Brontispa longissima Naturally Infected by Metarhizium anisopliae

[Objective] The purpose was to supply reference basis for studying invasion region of Metarhizium anisoplia in Brontispa longissima, invasion process and its control measures. [Method] The surface of B. longissima naturally infected by M. anisoplia which were collected from field were scanned and observed by scanning electron microscope. [Result] The regions naturally infected by M. anisoplia on surface of B. longissima included not only arthrodial membrane between abdomen and prothorax, but also both sides of abdomen and the rear part of end node of abdomen, where a large amount of structural material closely related to infection had been formed and observed, such as infection peg, mycelia and spore. [Conclusion] Damage degree of insect surface were different when M. anisopliae infected B. longissima under different environmental conditions, such as nutrient material, PH value, temperature and humidity, etc.

The production relationship of destruxins and blastospores of Metarhizium anisopliae with virulence against Plutella xylostella

Metarhizium anisopliae as an essential entomopathogenic fungus has been known to produce destruxins （a kind of cyclo-peptidic mycotoxins） and blastospores in submerged culture. Blastospores and destruxins are candidates for in- secticides, but the relations of both productions and the impact factors are unclear yet. In this study, we investigated the effects of inoculums, rotation, dissolved oxygen （DO） on the productions of blastospores and destruxins A and B （DA and DB） in submerged culture of M. anisopliae strain MaQ01. The results indicated that DO levels were regulated by inoculum amounts and rotation speeds, meanwhile, the productions of DA, DB and blastospores were also closely influenced by those factors. Totally, when DO value was more than 40%, the higher productions of destruxins and blastospores were achieved, by contrast, lower than 40% of DO values resulted in lower productions. The regression analysis suggested that the productions of DA, DB and blastospores were positively correlated with the DO levels. Meanwhile, the positive correlations between the productions of DA or DB and blastospores were also found. Briefly, when the rotation is 150 r min-1 and the inoculum is 1.0xl06 spore mL-1, the DA, DB and blastospores achieved the best production of 61.81 mg mL-1, 24.74 mg mL-1 and 5.73x 108 spore mL-1, respectively. In addition, the pathogenicities of blastospores and conidia against Plutella xylostella were bioassayed. The higher mortalities of P. xylostella were totally recorded in blastospore treatments than in conidia treatments, especially in lower dosages and earlier periods. Our research will give some new insights to production of destruxins and blastospores by using M. anisopliae.

Optimization of the Chitinase Production by Different Metarhizium anisopliae Strains under Solid-State Fermentation with Silkworm Chrysalis as Substrate Using CCRD

Entomopathogenic fungi, such as Metarhizium anisopliae, are able to control various insect pests. These fungi attack the integument of the host using an enzymatic complex. Among the enzymes found in this complex, chitinase is an important component. However, the relation between the chitinase production and the virulence from different M. anisopliae strains has not been analyzed. In this manuscript it is presented the chitinase production by four M. anisopliae strains with different potential of virulence in Solid-State Fermentation using silkworm chrysalis as substrate. The higher chitinase level was obtained with the strain IBCB 360 (7.14 U/g of substrate) with potential virulence of 68% on Diatrea saccharalis. The enzyme production was optimized for all strains using a factorial planning (CCRD) considering the cultivation time and medium humidity as independent variables. The maximal production of chitinase was obtained at a range from 8 to 12-days old cultures and from 45% to 62% of moisture according to the surface response plot, with high R2 value. The enzyme production by the strain IBCB 167 was increased two-folds under optimized conditions, while for the strains IBCB 360 and 425 the chitinase production was increased four-folds and nine-folds for the strain IBCB 384.

溶洞真菌Metarhizium anisopliae NHC-M3-2中两个新的异香豆素类化合物

对来自广西伊岭岩风景区喀斯特溶洞真菌Metarhizium anisopliae NHC-M3-2的次级代谢产物进行提取研究。利用薄层色谱、高效液相色谱等对真菌次级代谢产物进行分离纯化,得到10个化合物,其中7-羟基-3-羟甲基-5,6-二甲基异铬-1-酮(1)与7-羟基-3,5,6-三甲基异铬-1-酮(2)为新结构的异香豆素类化合物,N-乙酰苯丙氨酸(3)、毛壳素J(4)、2-one-13-hydroxy-3,5,8,7(11)-eudesmatetraen-12,8-olide(5)、1H-吲哚-3-甲醛(6)、irpexolaceus B(7)irlactinL(8)、细胞松弛素K(9)、烟曲霉酸(10),共8个已知化合物。运用核磁共振谱、质谱等方法确定化合物结构,分别采用CCK-8法对化合物进行肿瘤细胞毒活性评价,结果显示,新化合物2对HepG2细胞的IC_(50)为29.83μmol·L^(-1),化合物9对HCT116细胞的IC_(50)为27.44μmol·L^(-1)。

伊岭岩溶洞真菌Metarhizium anisopliae NHC-M3-2中一个新的萘并吡喃酮糖苷

利用硅胶和高效液相等柱色谱方法对溶洞真菌Metarhizium anisopliae NHC-M3-2发酵产物进行分离纯化,得到了7个化合物。通过紫外、红外、核磁共振谱与质谱方法分别鉴定为2,3-dehydroindigotide G(1)、(-)-regiolone(2)、萘吡喃酮(3)、indigotide G(4)、indigotide B(5)、破伤风毒素A(6)和破伤风毒素B(7),其中化合物1为新萘并吡喃酮糖苷类化合物。对分离得到的化合物进行抗乙型肝炎病毒(hepatitis B virus,HBV)活性测试,结果显示化合物3的抗HBV作用EC_(50)为4.5μmol·L^(-1),CC_(50)为92.3μmol·L^(-1),这是首次报道化合物3的抗病毒活性。

Trehalose and trehalose-hydrolyzing enzyme in the haemolymph of Locusta rnigratoria infected with Metarhizium anisopliae strain CQMa102

Topical application of the Metarhizium anisopliae var. acridum specialist strain CQMa 102 to the locust Locusta migratoria manilensis results in changes of the concentrations of trehalose and glucose in the haemolymph. Micrographs of the locust haemolymph shows Metarhizium anisopliae can effectivly penetrate the external skeleton of locust and after 2 days infection, the hyphae body will appear in the haemolymph of infected insects. The time in decrease of trehalose concentration coincided with that in increase of trehalose-hydrolysing enzyme activity in the haemolymph of the fungus-infected insects. Overlay gel analysis indicated there was considerably more trehalose-hydrolysing activity in the haemolymph of locusts infected by fungus than in controls. A comparable isoform was identified in in vitro culture of the fungus, suggesting a fungal origin for the in vivo enzyme. Haemolymph trehalose decreased significantly during mycosis of locusts by M. anisopliae. All these results suggested that this fungus may take advantage of competing nutrient utilization against the insect by its trehalose-hydrolyzing enzyme secretion. It may provide fundamental knowledge for fungal pathogenesis.

Plants Extracts and a Mycoinsecticide in Cowpea Yield Improvement in Guinean Savanah and Sahelian Savanah Agro-Ecological Zones of Cameroun

Cowpea yield improvement is done by adding agricultural inputs. The use of natural substances as pesticides is being encouraged to fight against cowpea field pests. The pesticidal potentials of Azadirachta indica and Boswellia dalzielii water extracts, Metarhizium anisopliae, alone and in combination with plant extracts, and a commercial synthetic pesticide (Decis®) were tested in field on two varieties of cowpeas, Vigna unguiculata in two agroecological zones (Guinean Savanah and Sahelian Savanah) of Cameroon. The field trials were carried out in a full randomized block design including nine treatments and a control. Four replications were made concerning the different treatments and control. At the flowering stage, the cowpea field was sprayed three times with different pesticidal formulations at the interval of five days. The number of ramifications per plant, and that of pods per block and seed yield were determined. The pesticide formulations considerably (p ®). Bafia variety treated with the combination of M. anisopliae and A. indica recorded the highest ramification rate (37.03 ± 1.59) in Maroua (Sahelian Savanah). The same variety also produced more important pods number (90.50 ± 16.66) in Ngaoundere (Guinean Savanah) with the binary combination of two plants used in this experiment. The highest seed yield (44.23 ± 2.31) was recorded in Ngaoundere with B125 variety treated with the combination of the three pesticidal formulations separately (A. indica, B. dalzielii, M. anisopliae). The plant products used in this work, M. anisopliae and their mixtures could supersede the synthetic pesticides considering environmental issue in cowpea crop protection. Then, such formulations would not only improve crop productivity but also preserve environment from the pollution due to the use of synthetic residual chemicals.

Entomopathogenic fungi disturbed the larval growth and feeding performance of Ocinara varians （Lepidoptera： Bombycidae）larvae

Feeding experiments using three strains ofentomopathogenic fungi, Beauveria bassiana, Metarhizium anisopliae and Isaria fumosorosea were conducted with newly moulted 3rd-5th instar Ocinara varians Walker larvae in the laboratory. The mortality of larvae immersed individually in spore suspension （1 × 10^7 spores/mL） of all the strains was 〉 80% except 5th instar larvae treated with M. anisopliae which transformed into pupae, but did not result in adult emergence. The growth （total body mass）, consumption, relative consumption rate and relative growth rate, were reduced at all three larval stages, while developmental time was extended in infected larvae with concurrent significant increase in approximate digestibility in infected larvae. Conversion of digested food （ECD） and ingested food （ECI） values declined in infected larvae as compared to the healthy larvae （control）. The 5th instar larvae treated with M. anisopliae showed higher ECD and ECI values than control. Based on mortality and growth inhibition it can be suggested that all the studied fungal strains have a high potential for biocontrol and could be developed into biocontrol agents against O. varians.