Screening and identification of antagonistic Chaetomium spp· against Monilinia fructigena

YUE Haimei; ZHUANG Hua; GONG Wenfeng; ZHANG Xinjun

doi:1O.13925/j.cnki.gsxb.20160422

Abstract：[Objective] Moniliniafructigena is a common disease in the late growth and storage period of apples.It can cause decay of apple fruit and directly affect its economic value.At present,chemical fungicides are used to prevent and cure them,and long-term use of chemical fungicide pathogens produce drug resistance,while pesticide residues will pollute the environment,break the ecological balance,and also affect human health.Chaetomium spp.is a type of fungus with biocontrol potential,and there is no biological control on the brown rot of apples.In order to enrich the biocontrol strains of postharvest diseases of apple trees in China,a series of experiments were carried out to isolate Chaetomium spp.from different substrates in Tibet.[Methods] Inhibitory effect of Chaetomium spp.against M.fructigena was determined by performing an intra-plate antagonistic test.The biocontrol of Chaetomium spp.and M.fructigena were inoculated on PDA plates,respectively.The biocontrol agent was located 4.5 cm apart from the pathogen.The growth radius of M.fructigena and the growth radius of M.fructigena fungus were treated respectively at a temperature of 28 ℃ for 10 days,and the inhibitory rate was calculated.The inhibitory rate of the crude extract of Chaetomium spp.on M.fructigena was determined by using the drilling method.Under aseptic condition,M.fructigena was inoculated in the center of the PDA plate,four holes were punched at a 4.5 cm distance from the pathogen using a 6 mm diameter sterile punch respectively,two holes into the 200 mg· L-1 crude wool crude extract of dilution,negative control solution or positive control solution of 150 μL,using the mycelial growth rate method,respectively.The antimicrobial activity of the crude extracts of Chaetomium spp.against M.fructigena was determined on the 5th day,the 7th day and the 9th day.The in vitro inoculation method was used to determine the control effect of the fermentation liquid of Chaetomium spp.on M.fructigena.The apples were neat and healthy.The surface was disinfected with 75％ alcohol,and sterile nails were used to evenly puncture 3 to 4 mm deep wounds on each apple,after 1 h we added 20 μL biocontrol bacteria fermentation broth,PDB liquid medium,50％ carbendazim solution,20 μL PDB liquid medium (negative control into the holes to verify the apple fruit' s condition without M.fructigena).Three hours later,20 μL of M.fructigena spore suspension with a concentration of 1 × 106 per mL was inserted into the three wells by pipetting.The negative control had no M.fructigena pathogen spore suspension,and was then dried and wrapped in polyethylene bags wrapped in moisture,stored at 25 ℃,7 d after measuring the lesion radius.The strains were inoculated on a cornmeal medium (CMA) and cultured in dark at 28 ℃ for 10 days,and the fruiting body was fully matured.The morphology of the ascus,the shape of the appendages,the morphology of the ascus,the morphology of the ascospores,the color,the position and the number of the germination holes were observed with a Leica microscope DM5000,and the morphology of the subcapsules were observed with a Nikon dissecting microscope.Photographs were taken,combined with colony characteristics,ascospores maturation time for measurement,recording and morphological identification.The genome of the strain was amplified by PCR using ITS-1F and ITS4 of the rDNA-ITS internal transcribed spacer (rDNA-ITS).The PCR reaction system was 25 μL:10 μL PCR buffer 2.5 μL,DNA template 15 ng,2.5 μL·L 1 dNTP 2.0 μL,1 μL each of 10 μmol·L-primer,0.2 μL of 5 U· μL-1 Taq enzyme,and finally double-distilled water was used to make up to 25 μL.35 ℃ for 30 s,35 ℃ for 30 cycles,and 10 min for 72 ℃.The homologous sequence was searched in GenBank by using Blast.The DNA-ITS sequences of 18 related species were cloned using CLUSTALX and constructed phylogenetic trees with MEGA 5.10 under 1 000 replicates (Melanocarpus thermophile was selected as outgroup).[Results] The antagonistic effect of Chaetomium sp.24-9 on the growth of M.fructigena was the best at the 3rd day,and the inhibition effect was the most obvious at the 7th to 9th days after culture.The inhibition rate to M.fructigena was 40.00％,and the inhibition band width to pathogen was 2.0 cm.The results showed that the inhibitory effect of 30 g· L 1 60％ carbendazim was 66.67％.The apple pieces treated with the fermentation liquid of Chaetomium sp.24-9 showed good control effects after 4 days of inoculation,and the inhibition rate to M.fructigena reached 62.18％.The other three strains of Chaetomium spp.in the control of M.fructigena showed no significant effects,with the inhibition rates below 30％.The results showed that the growth rate was 7-8 mm,and the aerial mycelium was sparse,with light olive color secretions sporule fruit surface health,spherical to obovate,diameter (232-304) μm× (261-350) μm,with a fixed orifice,ascomycetes began to mature after 7 d,the subcapsular fruit wall in the reflected light was light brown;the fruit wall cell polygonal shape was not corolla,with a separation,the base width of about 3.75 μm;subcapsular clavate,clustered,with stalk,apex acuminate,adaxially glabrous,and adaxially glabrous.Cysts spores were brown,lemon-shaped,smooth on both sides,and with both ends of the protrusions,(7.4-9.8) μm× (9.3-13.5) μm,there is an obvious terminal germination hole.The isolate was identified as C.globosum according to its morphological characteristics.DNA was extracted from C.globosum 24-9 and amplified with primers ITS-1F and ITS4 to obtain a target fragment of about 600 bp length.The nucleotide sequence of 567 bp was obtained by sequencing.The sequence was submitted to GenBank (accession number:KY132127).The phylogenetic relationship between C.globosum and Chaetomium sp.24-9 was confirmed by the homology alignment of the rDNA-ITS sequences of 18 related species.[Conclusion]The results indicated that Chaetomium sp.24-9,which had good antagonistic activity against M.fructigena,was identified by laboratory tests.The results indicated that the strain could beused as an effective material for apple prevention of postharvest diseases.