Evaluation Of The Potential Of Bipolaris Bicolor Strain SYNJC-2-2 As A Biocontrol Agent Of Weeds In Tea Gardens

Tea plant（Camellia sinensis（L.）O.Ktze）is an important economic crop due to the multiple utilization value.China is the earliest contry to cultivate tea plant in the world.China possesses a rich varieties of tea plant resources and the lagest areas of tea plant cultivation.Weed is one of the primary reasons that negative affect the yield and quality of tea.At present the management strategies of weeds in tea garden mainly include manual labor,chemical herbicide,physical and ecological ways.Recently,a higher standard of pesticide residue has been required when tea products are exported to the European Union.Thus,the application of chemical herbicides in tea garden is limited.It is imperative to develop a safety,environmentally-friendly and efficient bioherbicides applied in tea.This study aims to to screen out plant pathogens with strong pathogenicity to weeds and high safety to crops,and to evaluate the herbicidal potential,and provide candidate strains for future biological control.We investigated the main weed species from 16 tea gardens in Jiangsu,Zhejiang and Anhui provinces,and collected 114 samples of weeds.214 fungal strains were isolated from the samples and 44 kinds of plant pathogens were screened out.Thereinto,11 species of pathogenic fungi were found,which can lead to a large lesion with more than 10 mm of diameter formed on the detached leaves of the host weeds.According to the morphological comparison and DNA sequence analysis,these 11 strains were identified as Bipolaris bicolor,Bipolaris setariae,Alternaria alternata,Curvularia intermedia,Curvularia lunata,Fusarium proliferatum,Fusarium equiseti,Corynespora cassiicola and Didymella bellidis,respectively.Based on the reference including science reports and patents,considering crop safety,B.bicolor strain strain SYNJC-2-2 were selected.Results show the strain SYNJC-22 has highly herbicidal activity,both in vitro and in vivo.The application of 5×104 spore mL1 of B.bicolor strain SYNJC-2-2 exhibited above 95%control efficacy of Eleusine indica.The result suggest that B.bicolor strain SYNJC-2-2 is a candidate strain as a biocontrol agent of weed E.Indica.To eveluate herbicide activity and crop safety of B.bicolor strain SYNJC-2-2,17 crop species from 8 families and 42 weed species from 18 families were tested.The results show that B.bicolor strain SYNJC-2-2 can cause severe damage on grass weeds,including Eleusine indica,Setaria viridis,Pennisetum alopecuroides,Microstegium vimineum,Digitaria sanguinalis,Echinochloa colonum,Elymus dahuricus,Arthraxon Beauv and Lolium perenne,but not deleterious effect on Camellia sinensis,Oryza sativa,Triticum aestivum,Nicotiana tabacum,Sesamum indicum,Spinacia oleracea,Isatis tinctoria,Lonicera japonica,Brassica napus and Arachis hypogaea.Maize and soybean leaves show the serious damage after inoculation with high concentration（5×105 spore mL-1）of spore suspension of B.bicolor strain SYNJC-2-2.After spraying with 5×104 spore mL-1,the highest disease index is more than 90 for E.indica,Setaria viridis and Microstegium vimineum.The mycelia growth of B.bicolor strain SYNJC-2-2 is usually affected on the nutrient source,temperature,pH and other factors.For B.bicolor strain SYNJC-2-2 growth,the optimum medium is PSA medium,the temperature range is 25-30℃,the optimal carbon source is sucrose,the optimal nitrogen source is glycine and asparagine,the pH range is 5-8.The alkaline（pH=8）or 12 h light/12 h dark conditions is more suitable for the mycelia growth of B.bicolor strain SYNJC-2-2.The strain can produce a large number of spores in the agar medium with juices of E.indica leaves.For spore production,the optimal pH is 5,the optimal temperature is 30℃,and the optimal carbon source is lactose,the optimal nitrogen source is lysine.Ultraviolet or black light irradiation is helpful for spore production.The germination of the spore requires more than 90%humidity.The optimal carbon source is sucrose,lactose or mannitol.The optimal nitrogen source is ammonium sulfate,ammonium chloride or lysine.Neutrally condition（pH=7）,room temperature（25℃）or full sunlight is more appropriate for the germination of spore.However,the spores will be completely dead after the high temperature（51℃）treatment for ten minutes.Here,the process of the host weed infected by B.bicolor strain SYNJC-2-2 on was studied.It was found that the strain can quickly lead to cell death in the detached leaves of the E.indica.The spore germinated germ tube after 3 hpi（hours post inoculation）.The end of mycelium expanded to form appressorium after 6 hpi.The leaf epidermis of host weed was ruptured and showed visible damage symptom after 12 hpi.In addition,the strain can attach on stomata or intercellular spaces by forming apesporium.In summery,B.bicolor strain SYNJC-2-2 exhibits strong pathogenicity and high capacity of spore reproduction.Meanwhile,strain SYNJC-2-2 shows high safety to tea,traditional Chinese medicine,rice,wheat,tobacco,cotton,rape and other crops s.Therefore,B.bicolor strain SYNJC-2-2 has the potential to be developed into a bioherbicide for weed control in tea garden and traditional Chinese medicine filed.However,before successful commercialization there are lots of further studies,which include the mechanism of weed infection,development of excellent formulation,application in field and so on.