| Water hyacinth (Eichhornia crassipes (Mart.) Solms) is a native plant species in the neotropics and is one of the most damaging weeds in the world. It was first introduced to China as a forage and ornamental plant in the 1930s, but later it escaped from the introduced habitats and became one of the most important alien invasive plant species in China. At present, chemical control-herbicides are used to control water hyacinth in most countries. However, with the environmental protection consciousness strengthening, biological control (such as the uses of natural enemies, pathogens and plant allelopathy) was deployed as one of the effective and environmentally acceptable approaches. In this study, in 2009 and 2010, disease surveys were conducted on field water hyacinth plants in different areas of Chongqing. Two new diseases, showing brown spot and leaf spot and diagnosed following the Koch’s Postulate steps, were recorded. The pathogens were isolated and identified, as well as their major bio-characteristics.1. Brown spot of water hyacinthDisease iagnosis. Lasions on leaves of water hyacinth plants infected are oval or shuttle-shaped; they become strips later. Heavily diseased leaves be partially blighted or killed. Five pathogenic isolates were obtained from the diseased leaves and isolate WHO 10 was shown the most virulent to water hyacinth plants and was identified as the pathogen of the plant via Koch’s Postulate procedures; Strain WHO 10 induced brown spot and blight symptom on leaves of the weeds and the disease was named brown spot of water hyacinth based on the symptoms. The mean disease severity (DS) on leaves was up to 6.84 (Charudarran scale) in four weeks after inoculation with mycelium of WHO 10.Pathogne identification. The colony on PDA plate is nearly round, primarily white and changed to gray or yellow colored later because of the productions of pigments and pycnidia. The mycelia are hyaline or colorless, septate. Conidia are fusiform or clavate-shaped,14.2-28.5×4.1-7.8μm, straight or slightly curly. Each conidium has four septae dividing it into five cells; the three cells at the middle are olive-brown and cylindrical while the other two cells at the ends are colorless, subuliform and smaller than the middle cells. There are two or three (mostly three) colorless appendages on the top end and one appendage at the bottom end of the conidium. The acervuli of WH010 are cup-shaped, disperse or aggregated, grew underleath the epidermis first and then protuberated outside. The base of the acervulus is dark-brown and formed from thick-sheathed, spindle-shaped cells. The rDNA ITS andβ-tubulin sequence of strain WH010 was also obtained through PCR amplification (GenBank accession No JF502635 and JQ694097), BLAST analysis showed that WHO 10 was a species of the Pestalotiopsis genus and shared 99% of maximum identity with the species P. photinia.Major characteristics of strain WH010. One of the most optimum media for WHO 10 growth was potato dextrose agar (PDA), the most suitable temperature for colony growth was 25℃and the best acidity was pH6. Of the tested carbon and nitrogen resources, WHO 10 grew and sporulated normally on media contained glucose, maltose, sucrose, beef extract or peptone. The fungus produced significantly higher amount of spores on PDA. The suitable temperature for sporulation was at 22-28℃and the suitable pH value was pH5-10. Light was conducive to colony growth and sporulation, but would inhibit conidial germination. Normally, conidia of WHO 10 germinated at the range of pH4-12 but pH6 was most favorable for germination. The pathogenicity of both the fungus and its culture filtrate were examined on 21 common and important plants from eight different plant families and there was no disease symptom observed.2. Leaf spot of water hyacinthDisease iagnosis. The typical symptoms of the disease were characterized the initial small circular ink black spots on leaves, which developed to oval spots and may join together to form large irregular spots. The spots were black at the center, deep black at the border and surrounded by a yellow halo of newly-infected tissue. Ten pathogenic isolates were obtained from the diseased leaves and isolate WA10A was shown the most virulent to water hyacinth plants and was identified as the pathogen of the plant via Koch’s Postulate procedures; strain WA10A induced black spot and blight symptom on leaves of the weeds and the disease was named leaf spot of water hyacinth based on the symptoms.The mean disease severity (DS) on leaves was up to 8.98 (Charudarran scale) in four weeks after inoculation with mycelium of WA10A. All the inoculated leaves were killed within five weeks.Pathogne identification. On potato dextrose agar (PDA) plates, the WA10A fungus grew fast and is nearly round, primarily white and changed to apricot or red colored later because of the productions of pigments and pycnidia. The mycelia are hyaline or colorless or red, septate. The fungus was morphologically characterized by its reddish perithecia, color change in 3% potassium hydroxide (KOH), waited perithecial wall cells, and unitunicate asci with a long stalk and 1-3 septate ascospores. Asci were unitunicate, clavate, without a differentiated apex,300-400μm, and tapered to a long, thin stalk.Ascospores were aggregated in the upper third of the ascus and were fusoid with rounded ends, straight to slightly curved,1-to 3-septate, mostly 1-septate, colorless, and 38.5-49.5×4.1-5.8μm. The rDNA ITS andβ-tubulin sequence of strain WA10A was also obtained through PCR amplification (GenBank accession No JF502694 and JQ694096), BLAST analysis showed that WA10A was a species of the Calonectria genus and shared 99% of maximum identity with the species C. ilicicola.Major characteristics of strain WA10A. One of the most optimum media for WA10A growth was potato sucrose agar (PSA), the most suitable temperature for colony growth was 25℃, and colony growth at the range of pH5-10 but the best acidity was pH6. Of the tested carbon and nitrogen resources, WA10A grew and sporulated normally on media contained glucose, soluble starch, beef extract or peptone. The fungus produced significantly higher amount of spores on PDA or PSA containing spider lily leaves extract liquid. The best temperature for sporulation was at 28℃and best pH value was pH6. Conidia of WA10A germinated normally at 25-28℃. Light was conducive to colony growth and sporulation, but would inhibit of conidial germination. Using ethyl acetate to extract crude toxin of WA10A, and then, the oil phase of dichloromethane’s extraction was analyzed by column chromatography, eight bottles of the merger were separated. After the bioassay of the eight combined bottles, No.3,6 and 7 bottles were all found to be toxic to leaves. The results indicated that column chromatography separation of toxins played a better role, and the separation effect was obvious. The pathogenicity of both the fungus and its culture filtrates were examined on 21 common and important plants from eight different plant families. It only on Cucumis sativus and Benincasa Saviamong among the successfully-cultivated in the safety of 21 kinds of plants tested, and culture filtrates had no virulence on all plants.According to investigation, water hyacinth brown spot and leaf spot diseases were two commonly distributed diseases and of potential use in biocontrol for the plants. Two fungal pathogens were isolated from the diseased plants and identified as P. photinia and C. ilicicola, respectively. Major biological characteristics results indicated that P. photinia and C. ilicicola can adapt well to acid and high temperature, and maintain a higher ability of propagation and survival. P. photinia of water hyacinth has a high specialty, and the pathogen and its culture filtrates had no virulence in the safety of 21 kinds of plants tested. C. ilicicola of water hyacinth has a high specialty, it only on Cucumis sativus and Benincasa Saviamong the successfully-cultivated in the safety of 21 kinds of plants tested, and its culture filtrates had no virulence on all plants. All the researches above will lay the foundation for the two pathogens on water hyacinth biological control applications. |
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