| Mango(Mangifera indica)is the fifth fruit being widely produced in the world.Hainan province is the largest mango production area in China because of unique tropical climate and superior terrain,which is most suitable for flowering and growth of mango.Mango disease was nearly 30 kinds in the world in 1973,and about 20 kinds in China in 1988.However,mango disease reached 88 kinds in China in 2015.That showed a growth trend of mango disease in our country.Mango sooty blotch disease(Cladosporium cladosporioides,C.sphaerospermum)is one of new reported mango diseases during 2010-2015 years in China.In 2007,this disease was first discovered in Changjiang County in the west of Hainan province,and has been spreaded into Sanya,Ledong,Changjiang and Dongfang now.However,there are rarely reports about it.In this paper,to provide the theory basis for the early diagnosis of the disease,prediction and control in field,biological characteristics,parasitic site,rapid PCR detection system,GFP-marked C.cladosporioides,and screening of fungicide,antagonistic organism and foliar fertilizer were finished.The main results were as follow:1.Biological characteristics.For mycelial growth,the optimal medium was mango dextrose agar medium riching in organic nutrition.The optimum temperature was 20 ℃ and the optimum pH value was 7-8.The effect of light was not significant.The organic carbon source lactose or nitrogen source glycine was respectively the optimum carbon or nitrogen source.Lethal temperature of mycelium was 50 ℃ for 10 min.For sporulation,the optimum temperature was 25 ℃ and the optimum pH value was 6.0.Alternation of light and dark,maltose and L-acystine were favor to sporaluation.For spore germination,the optimum temperature was 25 ℃,the optimum pH value was 7.0,and the optimum RH was liquid water.Organic carbon source arabinose or nitrogen source L-asparaginate was favor to spore germination.Lethal temperature of spore was 50 ℃ for 10 min.2.Parasitic site.Mango branches,leaves,flowers,fruits and weeds in the mango orchard were preliminarily determined as the parasitic sites of C.cladosporioides by isolation and identification of the pathogens from different areas,plants,varieties and organs of mango.C.cladosporioides may infect cultivated variety of mango as Guifei,Jinhuang,Tainong,Jidan,and Jinhui.3.Rapid PCR detection system.Eight pairs of specific primers were designed by comparison and analysis of rDNA-ITS from different pathogens on mango.Two pairs of specific primers ML-SF9/SR5 and ML-SF10/SR10 were screened and amplification products were 408 bp and 424 bp in size,respectively.There are two groups of nested-PCR primers with ITS1/ITS4 as outer primer,and ML-SF10/SR10 and ML-SF9/SR5 as inner primers,respectively.Two nested-PCR systems could inspect C.cladosporioides from field mango fruits and their sensitivity of detecting pathogen DNA was as low as 35.5×10-10ng/μL.4.GFP marked C.cladosporioides.After gfp and hygB gene inserting to site-directed mutagenesis in ITS sequence,plasmid pITGH was successfully constructed.A transformants of C.cladosporioides with stable green fluorescence in hyphae,germinating conidia was gained by PEG-mediated method.There were no difference between the transformants and wild type in morphology,color,and virulence,but transformants was slower than the wild type strain in growth rate.The infection process of the GFP-marked strains indicated that C.cladosporioides only infected and developed on mango skin,but not in flesh of mango fruit.5.Screening of fungicides,antagonistic organisms,and foliar fertilizer.In the primary screening test,Pyraclostrobin,Polyoxin B,Carbendazim,Difenoconazole,Prochloraz,Difenoconazole-Propiconazole,and Bromothalonil have better inhibitory effect,EC50 values were between 0.17μg/mL and 0.59μg/mL.The next were Flusilazole and Propiconazole,EC50 values were between 0.73μg/mL and 0.80μg/mL.Trifloxystrobin-Tebuconazole and Tebuconazole were poor;EC50 values were close to 2μg/mL.C.cladosporioides was more sensitive to Carbendazim,Bromothalonil and Difenoconazole.One hundred and ninety-eight ratios from thirty-three compound agents were tested,and toxicity ratios of 26 ratios were greater than 1.2.Toxicity ratios were about 2.0 and showed the obvious synergy,when Pyraclostrobin and Propiconazole were mixed in the ratios of 1:4,and Prochloraz and Polyoxin B in the ratios of 2:3.Among of 14 antagonism strains,with better inhibitory effect of Bacillus spp.strains 5a-7 and 6c-5,as well as yeast strain M2,and the inhibition rates were respectively 79.80%,82.70%,and 46.00%.Among of 17 kinds of foliar fertilizer,the inhibitory effect of urea was best and with the inhibitory rate 72.5%,followed by boric acid,Plants power 2003+,Chitosan,Lufeng 95,ammonium sulfate,ammonium nitrate,and with the inhibitory rate of 43%-20.7%.Among of 12 combinations between foliar fertilizer and antagonistic organisms,the combination of Bacillus spp.strains 5a-7 or 6c-5 and urea or boric acid,and yeast strain M2 and urea combination showed good inhibition effects,and the inhibition rate were between 46%and 82%. |
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