Interaction Mechanism Of Aspergillus Welwitschiae Against The Root-knot Nematode Meloidogyne Graminicola In Rice

The root-knot nematode Meloidogyne graminicola is considered one of the most devastating pests in rice-producing areas and chemical nematicides are neither ecofriendly nor cost-effective.Thus,more acceptable biological agents are required for controlling this destructive pathogen.In the present research,a biocontrol fungus AW2017 was identified by morphological and molecular biological methods.By optimizing the fermentation conditions of nematocidal metabolites in 500 L fermentor,the optimum fermentation time of active metabolites was explored.The biocontrol potential of AW2017 to M.graminicola was preliminarily clarified in laboratory and greenhouse experiments.The defense mechanism of AW2017 against M.graminicola was further clarified by observing giant cell sections and analysis defense-related gene expression.Results will provide a new strategy for the green control of root-knot nematodes in rice.Based on morphological characteristics and molecular homology analysis,AW2017 was identified as Aspergillus welwitschiae,with a high similarity of 75%and 89%in Cam and BenA gene,respectively.In this study,the in vitro ovicidal and larvicidal activities of A.welwitschiae metabolites were tested on M.graminicola in laboratory experiments.The bioagent AW2017 displayed good nematicidal potential via its ovicidal and larvicidal action.The best larvicidal activity was observed at a concentration of 5×AW2017,which caused an 86.2%mortality rate at 48 h post inoculation.The highest ovicidal activity was recorded at a concentration of 5×AW2017,which resulted in an approximately47.3%reduction in egg hatching after 8 d compared to the control.The effect of A.welwitschiae on the attraction and infection of M.graminicola to rice was evaluated in a greenhouse.The attraction of M.graminicola to rice roots was significantly decreased in the AW2017 treatment.Inoculation of 2-week-old seedlings with A.welwitschiae reduced the attractiveness of rice roots to M.graminicola,resulting in a 37.3%and 39.6%reduction in juveniles migrating to 1.5-3.0 and 3.0-4.5 cm from rice roots,respectively,and a 64.9%reduction in the number of J2s that penetrated the roots.Under greenhouse conditions,the application of A.welwitschiae significantly reduced the root galls and nematodes in rice roots compared to the control.At a concentration of 5×AW2017,juveniles and root galls in rice roots at 14 dpi were reduced by 24.5%and40.5%,respectively.In addition,the development of nematodes in the AW2017-treated plants was slightly delayed compared with that in the PDB-treated control plants.Observing of giant cell sections under microscope showed that the surface area of giant cell decreased by77.6%than the PDB control and the cell development was significantly delayed.These results indicated that AW2017 can not only decrease the attractiveness of rice root to root-knot nematode but also reduce the infection of nematodes and delay their development.On the basis of the original shake flask fermentation,the fermentation condition for the production of the nematocidal metabolites in the 500 L fermentor was tested.Based on the pretreatment of the inoculation volume of 8%,the aeration volume of 4m~3/h and the dissolved oxygen level of 25%,the optimal fermentation time of AW2017 was determined to be 120 h.Under this condition,the corrected mortality nematode rate of AW2017 reachs as high as 91.3%compared with that in PDB-treated control.This fermentation will be useful in large-scale application of AW2017 in the future.Based on the qRT-PCR detection of related defense genes in rice roots,AW2017 could induce the up-regulation of ET synthesis-related gene expression after inoculated with M.graminicola.The relative expression of ET biosynthetic gene OsACS1 in the AW2017 treatment plants was significantly up-regulated at 72 h after inoculation,and the relative expression of ET biosynthetic gene OsACO7 was significantly up-regulated in the AW2017 treatment plants at 6 h after inoculation.However,no significant difference was observed in the relative expression of ET signaling pathway gene OsERF1,OsEBP89,OsERF70 and those tested genes in other signaling pathways.Results implied that AW2017might induced the express of ET synthsis-related genes to defense against the infection of root-knot nematode.In summary,A.welwitschia can inhibit the infection and development of rice root-knot nematode,induce the expression of defense-related gene and thus be a potential biological control agent against M.graminicola.The present research will represent a new strategy for development of biocontrol agent to effectively control of rice root-knot nematodes in the future.