Fengling GUO,Jinping WU,Zhixiong LIU,Zhaoyi DAI,Yunqiang WANG

Institute of Economic Crops,Hubei Academy of Agricultural Sciences,Wuhan 430064,China

Isolation and Identification of Main Fungal Pathogens in Muskmelon in Open Field of Hubei Province in Summer

Fengling GUO,Jinping WU,Zhixiong LIU,Zhaoyi DAI*,Yunqiang WANG

Institute of Economic Crops,Hubei Academy of Agricultural Sciences,Wuhan 430064,China

Fungal diseases often occur seriously in muskmelon in open field of Hubei Province in summer,especially in continuous cropping pattern,resulting in great economic losses.In this study,the pathogens of main fungal diseases in muskmelon in open field of Hubei Province were isolated,and they were identified by morphological and molecular techniques.The results showed that muskmelon fusarium wilt is a major disease in muskmelon in open field of Hubei Province in summer,and its pathogen was confirmed to be Fusarium oxysporum.In future studies,one pair of specific primers would be designed to detect different pathogenic races of Fusarium oxysporum so as to accelerate the detection and to shorten the detection time, thereby proving guidance for actual production.

Muskmelon;Fusarium wilt;Isolation and identification

I n 2013,the cultivation area of muskmelon in HubeiProvince was 22 500 hm2with a total output of about 716 900 t.The annual output value of muskmelon in Hubei Province is 1.449 billion yuan.But with the extension of continuous cropping,soilborne diseases occur more and more seriously in muskmelon,especially in open-field cultivation in hot and rainy summer.The loss caused by fungal diseases to muskmelon is generally more than 15%.In heavy disease area,the muskmelon plants in a field will almost die,and even get a harvest failure.To this end,the pathogens of fungal diseases were isolated from muskmelon growing areas where fungal diseases occurred more seriously, and they were identified by morphological and molecular techniques so as to provide guidance for diseases control in muskmelon cultivation.

Materials and Methods

Tested strain

The diseased muskmelon samples were collected from the muskmelon base in Hubei Academy of AgriculturalSciences,the muskmelon base of Hubei Academy of Agricultural Sciences in Jinshuizha and the muskmelon base of Wuhan Tianxiaxian Modern Agricultural Development Cooperatives.The pathogen was isolated by organizational separation[1], and then purified and preserved.

Morphology observation of pathogen

The pathogen was inoculated in the potato-sugar-agar(PSA)medium, and cultured at 28℃in dark for 3 d. The colony color and morphology were observed.The conidia were washed with sterile water,and their morphology was observed under a microscope (XDS-500 inverted microscope).

Biological property determination of pathogen

For the pH test,the medium pH value was designed as 5,6,7,8,9 and 10,respectively.A lawn in diameter of 5 mm was inoculated in the center of each PSA medium,and cultured at 28℃in dark for 3 d.After then,the colony diameters were measured using crossing method.For each medium,3 replicates were arranged.

For the lethal temperature test,a total of 5 temperature treatments(35, 40,45,50 and 55℃)were designed. There were 6 replicates foreach treatment.After heated in thermostatic water bath for 15 min,the lawns were rapidly cooled to room temperature. Subsequently,the lawns were inoculated in the center of the PSA plates, and cultured at(25±1)℃for 3 d.After then,the growth status of the mycelia was observed.

For nutritional conditions test,the basic medium adopted the Czapek medium,whichwascomposedof sodium nitrate (2 g),potassium chloride (0.5 g),ferrous sulfate (0.01 g), dipotassium phosphate (1.0 g),magnesium sulfate(0.5 g),sucrose(30 g) and agar(17 g).The original carbon sources in the basic medium were replaced by glucose,D-fructose,lactose, mannitol and maltose;and the original nitrogen sources were replaced by glycine,yeast extract,ammonium sulfate,potassium nitrate,peptone,ammonium dihydrogen phosphate,ammonium nitrate and L-glutamate.The incubation was performed at 28℃for 3 d.The colony diameters were measured using crossing method,and there were 3 replicates for each treatment.

18S rDNA amplification and sequence analysis of pathogen

The pathogen lawn was inoculated in the center of the PSA plate and cultured at 25℃in dark for 48-72 h. The mycelia on the edge of the colony were cut into pieces in diameter of 2-3 mm.Total 4-5 mycelia pieces were inoculated in the PSA liquid medium and cultured in a thermostatic oscillation incubator(28℃,180 r/min)for 2 d.Sub-sequently,the broth was filtered through a double-layer nylon web to collect the mycelia, which were washed with water twice to remove the culture medium inside.The collected mycelia were wrapped in silicone overnight to drain water.Then,the mycelia were ground into powder in liquid nitrogen,and the mycelial NDA was extracted with CTAB method[2].

The primers ITS-F and ITS-R were adopted for amplifying the ITS region of pathogen 16 S rDNA.Their sequences were as follows[3]:18S-1 (5’-GTAGTCATATGCTTGTCTC-3’); 18S-2(5’-TCCGCAGGTTCACCTACGGA-3’).

The PCR reaction system was as follows:triphosphate deoxynucleotide (10 mmol/L)0.6 μl,magnesium chloride (20 mmol/L)1.2 μl,buffer 2.0 μl, ITS-F (10 μmol/L)1.0 μl,ITS-R(10 μmol/L)1.0 μl,Taq polymerase(2 U/ml) 1.0 μl,template 0.1 μl,ultra-pure water 13.1 μl.

The PCR reaction conditions were as follows:pre-denaturation at 94℃for 2 min;denaturation at 94℃for 30 s,annealing at 45℃for 30 s,extension at 72℃for 2 min,25 cycles;extension at 72℃ for 5 min.The PCR products were examined by 1%agarose gel electrophoresis.The results showed that a 1 800-bp band was shown on the electrophorogram.The PCR products were recovered,and the recovered cloning fragment was ligated with pGEM-T vector,which was then transformed into E.coli DH-5α competent cells.Subsequently,the blue-white screen was performed,and the positive colonies were proliferated. The broth was sampled and submitted to PCR using M13.The PCR products were examined by agar gel electrophoresis.Then the positive colonies containing the target fragment were sequenced (Beijing Genomics Institute).The sequencing results were submitted to GenBank for analysis. According to the sequencing and morphological analysis results,the isolated pathogen was identified.

Results and Analysis

Disease symptoms and pathogen morphology and pathogenicity

As shown in Fig.1,the vine bases of muskmelon were constricted slightly,and their skin was rough.The rotting lesions on muskmelon fruits were water-soaked,and there were white or pink mold-shaped substances on their surface.Fig.2 showed the morphology of isolated conidia of the pathogen. The conidia were unicellular and ovoid. Fig.3 showed the symptoms in muskmelon when the pathogen was back inoculated.The symptoms were similar to those in muskmelon in field.

Biological properties of pathogen

The pathogen could all survive in the pH range of 5-10.It showed the best growth in the culture medium at pH 7.0.The mycelia grew rapidly and were dense and compacted.It suggested that a neutral medium was more suitable for the growth of pathogenic fungi(Fig.4).The lethal temperature of the pathogen was 50℃.As shown in Fig.5 and Fig.6,the effects of different nutritional conditions on growth of pathogenic fungi in muskmelon differed greatly.Considering both mycelial expanding and growth,the optimum carbon sources forgrowth ofthe pathogen were soluble starch and lactose,and the optimum nitrogen source was yeast extract.

18S rDNA sequence verification of pathogen

Using primers 18S-1 and 18S-2,a fragment in a size of about 1 700 bp was amplified from the genomic DNA of the pathogen.The sequencing results showed that the18S rDNA region of the pathogen had a size of 1 720 bp. The obtained sequence was submitted to GenBank BLAST.The results showed that the obtained sequence has a homology of 99%with Fusarium oxysporum.In addition,phylogenic tree was constructed.The pathogen was also divided into the same group with Fusarium oxysporum,which further confirmed that the pathogen was Fusarium oxysporum(Fig.7).Fusarium oxysporum is the pathogen of muskmelon Fusarium wilt.

Conclusions

Fusarium wilt can occur in the whole growth period of muskmelon, but it mainly occurs in the middle and later growth period.Its transmission media are mainly soil and plant debris. Seeds can also carry pathogen.Due to hot and rainy summer in Hubei Province,Fusarium wilt occurs more seriously in muskmelon in open filed. There have been many reports on pathogen,pathogenesis law and control techniques of muskmelon Fusarium wilt[4].But there has been no report on muskmelon Fusarium wilt that occurs in Hubei Province.Fusarium wilt has a variety of pathogenic races.In this study,the pathogen of muskmelon fusarium wilt is confirmed as Fusarium oxysporum by morphologicaland molecular techniques,but its specific physiological race has not been identified.Therefore,in future studies,one pair of specific primers will be designed for identifying different pathogenic races of Fusarium oxysporum so as to accelerate the detection and to shorten the detection time, thereby providing guidance for actual production.

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Responsible editor:Tingting XU

Responsible proofreader:Xiaoyan WU

Supported by Earmarked Fund for China Agriculture Research System(CARS-26-34).

*Corresponding author.E-mail:guofenglingok@163.com

Received:April 2,2015 Accepted:July 6,2015