| As chemical pesticides have resulted in serious pollution, biopesticides have attracted more and more attentions. But, control effects of biopesticides were slow, which bring great risk to agriculture. Consequently, employment of biopesticides with chemical pesticides has become a new pathway in plant disease control. Trichoderma harzianum is an excellent biocontrol agent against a range of fungal plant pathogens. Because of its sensitive to benzimidazole fungicides, it's difficult to work with the chemicals in plant disease integrated control program. Therefore, to obtain excellent strains resistant to these fungicides and research their cooperations in disease control would appear significant for the exploration and application of novel biocontrol agent and chemical mixtures.α,β1,β2 andγtubulin gene were cloned from T. harzianum by degenerate PCR and inverse PCR. The results of BLASTP alignments showed that the four predicted proteins show high degree of homology with other fungal tubulins. T. harzianumα,β1,β2 andγtubulin amino acid sequences are found with similarities of 93%, 92%, 92% and 90% with the corresponding sequences of Neurospora crassa. The positions of the conserved domains and motifs were identified. Simultaneity, the secondary structures and tertiary structures were predicted.In order to construct biocontrol strain with carbendazim resistance, BenR gene from N. crassa was specifically integrated intoβ2 tubulin site of T. harzianum using PEG-CaCl2 method. Southern blot analysis showed that the BenR gene was integrated into the genome of T. harzianum and the transformants were all genetically stable. Sensitivity tests showed that transformants can grow at 1500μg/mL carbendazim. The effective concentrations were up to 471.26μg/mL for mycelial growth and 307.21μg/mL for conidial germination, which appear 1200-fold higher than those of sensitive strain.Main biological characteristics of T. harzianum resistant strains were studied. The results showed that optimal temperatures for mycelial growth, conidial production and conidial germination are 25 to 30℃. pH values are not crucial, but slight acidity is beneficial to mycelial growth, conidial production and conidial germination. Light can stimulate conidial production significantly. Each resistant strains can growth, sporulation and germination at tested carbendazim concentrations, though biomass, sporulation and germination rate decreased. The optimal sporulation of resistant strain TS1 can be obtained on culture media of bran and straw at ratio of 2 to 1 with 55% water content, 25% inoculum sizes and temperature of 26℃. Under the optimal conditions, the production of conidia reached a concentration of 15.42×108 spores/g. Resistant strains appear positive cross-resistance to triabendazole, benomyl, and thiophanate-methyl.Antagonism and indoor control effect were studied. The results showed that T. harzianum resistant strains were proposed with broad-spectrum antifungal activities, which displayed strong vitro antagonistic abilities against eight plant pathogens tested. The inhibitory rate of resistant strain TS1 against plant pathogen Fusarium oxysporum and Rhizoctonia solani were 81.34% and 86.19% respectively. Control efficiency of resistant strain TS1 and carbendazim, in combination, was 82.22% against rice seedling blight, and 20.3% and 48.9% higher than treated groups single with TS1 and carbendazim respectively.Systemic induction of biocontrol fungi on main defense enzymes in rice seeding roots was studied. The results showed that the activities of POD, PAL, PPO and SOD of roots treated by strain TS1 and carbendazim in combination were higher than that in control. Except for SOD, the activities of defense enzymes treated by combination were not different significantly with that treated by strain TS1 only. Results indicated that combination can stimulate rice defense reaction, induce systemic resistance, but there is no synergistic effects were observed. Compared with T. harzianum T88, inducing duration of POD, PAL and PPO in rice seedling roots treated by strain TS1 were prolonged.Field control efficacies of resistant strain TS1 and carbendazim in combination against rice seedling blight were studied, and colonization ability of strain TS1 was conducted. The results showed that combination at three ratios all have synergistic effects on disease control, but the degree of synergism was distinct at different ratios. Significant synergistic effect could be found took place at ratio of 6 to 4, whose control efficiency against plant pathogen F. oxysporum and R. solani were 87.80% and 84.08% respectively. Strain TS1 was found can colonize on rice rhizosphere and rhizoplane, but carbendazim have no significant effect on colonization ability in sterilized soil. |
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