Development Of A Quantitative Detection Method For Metarhizium Anisopliae In The Mycoses Locust Using Real-Time Fluorescent PCR

Entomopathogenic fungi play an important role in bio-control. For its large host-range which includes many kinds of lepidoptera and its large scale use for bio-control, Metarhizium anisopliae have been one of bio-pesticides for using widely. But there are some problem existed in mycoinsecticide on the difficulty of strain breed, decline of virulence, lag in research of process and lack of quality control system, which inhibit the formation and development of the industry about the mycoinsecticide, the reason is mainly lied in the lack of methods on quick and exact assessment of virulence. The quick proliferation of pathogenic fungi in host is preliminary of death of host insects in despite of the very complex mechanisms in pathology, and the proliferating quantity and velocity affect directly to the pathogenic process and the pesticidal efficiency. It will meet the needs for strain breed, research and quality assessment of preparation and virulence assessment during supervising to set up the methods of quantified determination to the early infecting stage of pathogenic fungi in insects, which will help to find out the infecting speed and trend of pathogenic fungi. Moreover, the methods will supply technologic support to the production, research and determination of bio-pesticides in China. Our study includes, a) To extract the genomic DNA of M. anisopliae which can be used for amplification by PCR. b) To set up the determination system by fluorescent quantitative PCR to the M. anisopliae in locusts. The main conclusion is as follows: 1. The infected process of CQMa102 into Locusta migratoria manilensis was observed with a digital microscope. After the inoculation of M. anisopliae, hyphal body, which began to appear at 72 hours, could not be found in the haemolymph of locusts within 60 hours. From 72 hours to 96 hours, the hyphal bodies increased gradually in haemolymph, and at the antemortem time of locust, the haemolymph in which blood cells could be found hardly was full of M. anisopliae. 2. Primers which have high specificity were designed. Based on the sequences of the internal transcribed spacers(ITS) of ribosome DNA(rDNA) and 5.8s gene, the specific primers, which were suitable to the determination of M. anisopliae in locust by fluorescent quantitative PCR which used SYBR GREEN I as fluorescent material, were designed to distinguish between the genomic DNA of L. migratoria and M. anisopliae. 3. Standard curve was established for quantitative determination. After amplification by PCR with multiplicate reagents, the best reagent was found to be iQTMSYBR Green supermix of Bio-rad based on the relativity of standard curve and amplification efficiency of PCR. By use of the reagent, the amplification by PCR was done with 10 times diluted DNA of M. anisopliae as template, and the standard curve was got. The qualified range reached to 6 quantitative degrees (from 1x10-7 μg to 10-2 μg of DNA of M. anisopliae), and at least 10-7μg DNA in a 25μl mixture could be detected quatificationally. 4. The methods by which DNA of M. anisopliae could be extracted quickly from the haemolymph of infected locust were optimized. Pre-treated by 2-mercaptoethanol and digested by two enzyme, the cell wall of M. anisopliae could be broken drastically and quickly(less than 2 hours). By the filtration of microspin cloumn,the inhibitors of PCR were got rid off and the DNA templates used to the quantitative detect by PCR were obtained. The stability of the methods was tested. The value 0.007 of differential coefficient during five times repeats proved the methods were stabilized well. 5. The effect of the weight of the locust for test on the results of PCR. It was indicated that the weight of locust affected slimly to the PCR results if the weight range is within 0.6-0.8g. However, more than 80% of healthy male of locusts belonged to the weight range. 6. The effect of the instar of the locusts on the results of PCR. We proved that the instar affected strongly to the test results. Although it is easy for the locusts post eclosion 12 hours to be infected by M. anisopliae and the Ct value is small, the objection is the great difference among anits and the poor stability of test results. The results to the locusts post eclosion 36 hours were steady, but the speed of infection is low and the Ct value is large. Considering of the sensitivity and stability, we thought the best for quantitative determination is the healthy male of locusts post eclosion 24 hours. 7. Detected the growth dynamic process of M. anisopliae living in locust body. The result showed that M. anisopliae could be detected from locust heamlymph after inoculated for 36hrs (ahead of 30 hrs compared with traditional method). But there are less quantities of M. anisopliae which is out of lower limit for quantity test. quantity test can be carried out after inoculated 60 hrs. 60 hrs later, DNA concentration of M. anisopliae was up rapidly, DNA concentration of M. anisopliae at 72 is 1000 times than that of at 60 hrs, which is indicated that there are also Lag phase and exponential phasefor COMa102 when it grow in locust body. Conclusion: we established a method to detect M. anisopliae in locust by real time PCR. This method is quick, accurate and sensitive.