Endophytism And Growth-promoting Of Metarhizium Anisopliae During Peanut Development

Metarhizium anisopliae could interact with plants when it was used for pest control as a biocontrol agent. It has been reported promoting the growth of some plants and being an endophyte. The aim of this paper is to explore the effect of applied M. anisopliae on the peanut development and endophytism. The experiments included evaluating early development and whole development of peanut in M. anisopliae application, detecting the endophytism of this fungus in peanut root, and constructing egfp engineered strain for confirming the endophytic colonization and location. The results were as follows:1. The effect of M. anisopliae on the seed germination and the early development of peanut roots. Two inoculating methods, conidia-suspension-drenching and conidia-coating, were used when sowing peanut. Seed germination in 3d, taproot length and lateral root number in 16 d after sowing were detected. Meanwhile, the persistent population of M. anisopliae was monitored. M. anisopliae had no impact on the seed germination(P>0.05). The taproot length and lateral root number in conidia-suspension-drenching treatment both significantly increased compared to those of the untreated control in 4-10 d after treatment(P<0.05), whereas no significant difference was found between conidia-coating treatment and control in 16 d. The population dynamics of M. anisopliae in peanut rhizosphere soil of two inoculation treatments presented a similar trend of fluctuation in the first 8 d followed by a gradual decline. A larger decline appeared in the successive 8-12 d with a declining slope of 0.268 for conidia-suspension-drenching and 0.6378 for conidia-coating. A Pearson correlation analysis indicated that the ratio of the taproot length or lateral root number in conidia-suspension-drenching treatment and control was significantly relative to the persistent quantities of M. anisopliae(P=0.0216, R=0.8273 for taproot length; P=0.0032, R=0.9213 for lateral root number). This study concluded that M. anisopliae could promote the early development of peanut roots, and compared to conidia-coating inoculation, conidia-suspension-drenching was a more preferable inoculation method.2. The effect of M. anisopliae on the whole growth period of peanut. Two inoculating stages, sowing stage and pod-flower stage, were designed. The 11 parameters of peanut development and persistent population of M. anisopliae in peanut rhizosphere were investigated per 15 d. The results showed that the number of leaves and peggings, intact fruits, the wet and dry weight of peanut shoot, root and fruits in pod-flower stage treatment significantly increased compared to those of the control(P<0.05), whereas no significant difference was found between sowing stage inoculation and the control(P>0.05). The persistent population of M. anisopliae of pod flower stage treatment showed a higher colonization rate and proliferation rate than that of sowing stage treatment. The peak population of M. anisopliae in seedling stage treatment was 1.46 times than its original amount, this peak period coincided with the peak of white grub population, which might contribute to a high control effect. Nevertheless, the peak period of M.anisopliae in sowing stage treatment, keeping 0.69 times higher than its original amount, staggered the peak of white grub population resulting in a lower control effect than pod-flower stage treatment. Therefore, M.anisopliae could promote the growth of peanut, and pod-flower stage inoculating was a better occasion for peanut development as well as white grub control.3. Detection of M. anisopliae endophytism in peanut root. According to ITS sequence and marker gene sequence of the strain, two pairs of specific primers were obtained by design and screening. Their detecting sensitivity to target strain got to 1: 2000 and 1: 1000 in mixed DNA. Detection of inoculated root samples by PCR with the two pair primers and selective isolation obtained specific DNA fragments and target colonies from the samples of 75 d and 90d-old-roots in pod-flower stage treatment. It proved Metarhizium endophytic colonization in peanut root.4. Construction of M. anisopliae egfp engineered strain. Expression vector Camben-e GFP was constructed containing report gene egfp, resistant marker ben A3 gene. It was transformed into M. anisopliae M202 by Agrobacterium-mediated method. The positive transformants were examined by PCR and fluorescence microscopy, resulting in that transformants appear egfp gene fragments and green fluorescence. It proved egfp gene had been successfully transformed and expressed. These egfp engineered strain could be useful to further determine the colonization and localization of M. anisopliae in peanut.