Screening For Virulent Strains Of Metarhizium Anisopliae Against Monochamus Alternatus And Their Survival Dynamics In Forest

鈽匨onochamus alternatus is a major insect vector transmitting pinewood nematode disease, an important quarantine disease of pine forest. In this thesis, Metarhizium anisopliae strains virulent to M. alternatus were screened for and their survival dynamics in forest was studied. The results may provide with biological resources and theoretical basis to control M. alternatus.鈽匱hree hundred and thirty soil samples were collected from 110 forest fields, in which 88 were pine forest fields from 40 counties,cities or districts in Fujian province, 2 cities including Jinggangshan and Ji'an in Jiangxi province. Among these samples, M. anisopliae were detected by selective medium in 26 soil samples from 21 forest fields, of which 16 were pine forest, accounting for 7．9% of the sample and 19．1% of the fields. The isolation frequency of M. anisopliae ranged from 500 to 72500 colony forming units per gram soil (CFUè·¯g^-1 ), indicating its wide distribution in the forest soil.鈽匓ased on sporulation and germination rate from 20 different hosts from domestic and abroad, then their pathogenicity to M. alternatus were determined by using larva(3锝? intars)dip method (1脳10⁷ sporeè·¯mL) and adult-conidial attaching method. The result showed that there was significant difference in sporulation abilitiy, germination rate and pathogenicity betweeen different strains. Strains MaYTTR-04, MaYTTR-03, MaZPTR-01, MaJGSTR-01, JPMal291, JPMal349 and JPMa2049 were better, especially for strains MaYTTR-04, MaYTTR-03 and JPMa2049 with sporulation reaching (2．18卤0．12)脳l0⁸ sporesè·¯cm^-2 , (1．25卤0．0．05)脳l0⁸ sporesè·¯cm^-2 and (8．92卤0．16)脳l0⁷ sporesè·¯cm^-2 respectively. The summit of larva death was at 15 to 18 days post inoculation (dpi), and the cadaver rates at 21 dpi were 80%, 76．0% and 93．1%) respectively. The summit of adult death was at 12 to 15 dpi, and the adjusted mortality at 21dpi was 93．3% with 1．2脳l0卤2．0脳l0 spores/adult, 86．7% with 1．5×l0±5．0×l0 spores/adult and 94．4% with 3．4×10±3．24×l0 spores /adult respectively. The cadaver rates were 83．4%, 88．9% and 75．0% respectively, and the secondary degree cadaver,in which the area covered by conidia or hypha of M. anisoplia more than 50% of the cadaver's surface, were 66．7%, 55．6% and 53．1% respectively.At 25±1℃, the LT50 was 14．7 and 18．5 dpi when applying the amount of inoculum at 2．3×l0±0．2×l0 spores/adult and 2．3×l0±0．2×l0 spores/adult of MaYTTR-04 respectively. At room temperature (25～32℃), the LT were 12．9 dpi and 17．9 dpi respectively. Our data indicated that the strain was more virulent at higher temperature. When the adults of M. alternatus were kept in the cage in natural pine forest, average mortality was 60%) at 21dpi, and the cadaver rate was 48．9%.In order to study the survival dynamics of M. anisopliae in forest, strains JPMal349 and JPMa2049 were placed in pines bark of different direction; the spores of strains JP1349, JP2049 and MaZPTR-01 were blended with soil, then they were embedded into the soils of pure forest of Pinus massoniana , mixed forest of P. massoniana and Schima superba and gap of P. massoniana pure forest. And adult cadaver of M. alternatus infected by MaYTTR-04 were placed on the soil surface of mixed forest of P. massoniana and Schima superba.The spore germination rate of strains JPMa1349 and JPMa2049 which were set up in the bark of pine tree were more than 70% at 168d, yielding point of spore germination rate was from 31/01/2005 to 18/07/2005．There was certain difference in spore germination rate in different direction of the same strain. The time of spore germination rate at yielding point was longer in East with 196d and 168d for strains, and shorter in West with 163 d and 147d for strains JPMa1349 and JPMa2049 respectively . When spore germination rate was over 70%, the average time of yielding point of these two strains were 166d and 157d, however there was no statistically significant difference. At early stage, spore germination rate in different directions had no statistically significant difference, the standard deviation were less than 10%; at later stage, there was significant difference. Temperature was one of major factors which affected spore germination rate. Spore germination rate in the pine bark decreased when the temperature increased. Spore germination rate decreased rapidly in May, and it decreased most rapidly in June and July. Spore germination rate decreased rapidly if the average temperature higher than 28鈩? The survival dynamics of spore germination rate of M. anisopliae and Beauveria bassiana in pine bark was the same, and there was no significant difference between them at the same period鈽匒dult cadavers of M. alternatus which sporulation was 2．01脳10⁸ spores per adult were set on the surface of forest soil. The spore amount on cadavers reduced by 2．14times in 42d, it meant average amount of spore was 7．01脳10⁷ per adult. The spore amount on cadavers further reduced to 5．02脳10⁶ per adult after 84d, which was the same level as inoculum in the adult-conidial attaching method. The spore germination rate on every cadaver had significant difference in 126d, but average germination rates were all over 90%. When the larva of M. alternatus inoculated with spores from cadaver at the concentration of inoculation was 10⁶ spores per milliliter, the adjusted mortality was 45．6%.鈽匱he germination rates of strains JPMa1349 and MaZPTR-01 in different types of forest soil were further studied. The results showed that germination rates of tested strains were always more than 70% in two years. The amount of spore decreased to 27．8% and 51．4% of the original amount at 29d, but the amount of spore remained at more than 10⁷ spores per gram soil in 323 days; and lethal rate and cadaver rate of larva of M. alternatus were over 55% and 45% in 211 d.鈽匲tility model patent of automatic phoretic device(number of patent: ZL2005 2 0095343．7) of insect pathogen was approved. The device was assembled by upper traps and lower phoretic part. The inside wall of phoretic device had annulus with pathogen,e.g. M. anisopliae and B. bassiana. When the insect escaped from the device through the annulus, pathogen would be adhered to the insect and dispersed with the insect. Pathogen could be transmitted into stem by stem insects such as Dendroctonus spp., it could improve the infection chances for the larva which lived in the stem (such as larva of M. alternatus). In the field where phoretic devices were suspended, larva cadaver rate of M. alternatus infected by M. anisopliae was 32．1% higher than that in control field. The device could be applied in pest control in agriculture and forestry.Genetic diversity of M. anisopliae was analyzed using inter-simple sequence repeat (ISSR). Eighty-one fragments were amplified using 7 primers, in which 76 were polymorphic with 11．6 bands scored per primer. Analysis with program POPGEN revealed that there were certain genetic diversity between different population of M. anisopliae and in same population of M. anisopliae. UPGMA clustering analysis further showed that genetic diversity of M. anisopliae had certain corrrelation with geographical origin. Genetic variation of individual M. anisopliae was also significant on DNA level, suggesting that genetic differentiation occurred in M. anisopliae.In a summary, M. anisopliae is high virulent to M. alternatus, it could survive in pine forest and maintain bioactivity for a long time. The strains, MaYTTR-04,MaYTTR-03 and JPMa2049 may serve as a potential source of biocontrol agents for M. alternatus. The strain MaYTTR-04 could be used as industrial strain for mass production.