| Conyza sumatrens (Asteraceae), native to North and South America, is an important invasive, noxious weed and distributed widely in southern and southeastern China. It invades farm land and causes great losses to dry land crops, including wheat, corn, vegetables and beans. It also reduces biological diversity by crowding out native plants in the infested areas. There are different strategies (including mechanical or chemical technology and classical biological control) for controlling C. sumatrens in China. However, more effective and economical control strategies need to be developed for C. sumatrens weed management in different habitats. During a survey for fungal pathogens that could serve as potential biological control agents towards C. sumatrensis, a leaf spot disease of C. sumatrensis was observed in 2010 in Chongqing, China. Consequently, the objectives of the present study were to identify the disease, to isolate the pathogen and to study the potential of the pathogen as a biocontrol agent against C. sumatrensis.Symptoms of the leaf spot and isolation of pathogen. Typical symptom of this disease was purple spots with small circular on leaves and then developed to oval spots and got together to form large irregular spots. Ten isolartes were obtained from the diseased leaves. All of them were showed pathogenic to C. sumatrensis following the Koch’s postulate. A fungal isolate SMBC-22 with high virulence and infectiveness was selected as biological control agent agaist C. sumatrensis.Morphological and molecular identification of pathogenic strain SMBC-22. Colony of SMBC-22 on potato dextrose agar (PDA) plates was white initially and turned dark gray later. Colony was circular with smooth edge and obvious rings on the surface. Hyphae were septate, colorless or transparent, measured 1.2-2.4μm in diameter. Aerial hyphae were short but dense. Pycnidia were visible after 8 days of incubation, and they were black and immersed or semi-immersed on the medium. Pycnidia were measured 72 to 140μm in diameter. Conidia were produced in the pycnidia and they were hyaline, unicellular, ellipsoidal, and measured 4.5-5.8μm×1.6-2.1μm.To confirm identification of the fungus, genomic DNA was extracted from mycelia of SMBC-22. The internal transcribed spacer (ITS) gene of rDNA was amplified using the primers ITS4/ITS5. The ITS sequence was 524 bp in length and was registered in NCBI Genbank (Accession No. HQ645974).The LSU (large subunit) gene of rDNA was amplified using the primers LROR/LR7 and its sequence was 1300 bp (Accession No. JF966194). BLAST analysis showed that the present sequence had 99%homology to an isolate of Phoma macrostoma (DQ 404792 and GU238096) from Cirsium arvense (Canada thistle) in Canada. P. macrostoma was reported to cause chlorotic symptoms on its host plant. So SMBC-22 was identified as P. Macrostoma.Factors affecting growth and reproduction of SMBC-22. The most optimum medium for SMBC-22 growth was potato dextrose agar (PDA).The favorable temperature for the mycelium was between 20-30℃.The most suitable temperature for sporulation was at 25℃. pH 6 was the most optimum for its mycelium growth. Conidia can germinate well in the ranged of pH 5-10, and pH 8 is the best condition for germination. Light treatment have no obvious effect to sporulation and the thermal lethal point of this fungus was at 60℃for 20 min. Of the tested carbon and nitrogen resources, the fungus grew normally on media with glucose, sucrose, yeast ectract and NH4NO3. PDA medium was the best medium for sporulation of SMBC-22. The relative humidity (RH) for the condial germination must be above 95%.Host range of SMBC-22. The pathogenicity of SMB-22 on eighteen species of common crop plants within six families was tested. They were rice (Oryza sativa), eggplant(Solanum melongena L.), chillie(chili pepper), vax-gourd(Benincasa Savi), cole (Brassica oleracea), wheat (Triticum aestivum), corn (Zea mays), tomato(Lycopersicon esculentun), Chinese cabbage (Brassica campestris sp. pekinensis), green bean (Phaseolus vulgaris), rape (Brassica chinensis), turnip (Raphanus sativus), cowpea (Vigna unguiculata), spinach (Spinacia oleracea), cucumber (Cucumis sativus), squash (Cucurbita moschata), watermelon (Citrullus lanatus), suakwa (Luffa cylindrica). The results showed these crops were not infected by the pathogenic fungus (SMBC-22) 15 days after being inoculated. These results suggest that SMBC-22 may be used safely to control tumatre weed without damage to these economically important crops.Bioassay of toxin extracted from strain SMBC-22. Toxin of p. macrostoma was obtained by extraction with ethyl acetate. Pathogenicity of toxin was detected through bioassays on C. sumatrens leaves. Results showed that sympotoms on C. sumatrens leaves were observed under different pH values from 5.0-11.0 and the symptom was the same as that caused by SMBC-22 fungus. The txoin extract remained active after being treated at≤90℃for 1 hour.A common disease of invasive alien weed C. sumatrensis was investigated in Chongqing, China and a fungal strain SMBC-22 was isolated. The fungus was identified as P. Macrostoma according to its morphological characteristics and molecular method. The experimental results indicated P. Macrostoma was no infected to the main economic crops, so this fungus had been shown potentiallly valuable in C. sumatrensis bioloical control. |
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