Date of Award

2008

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Environmental Science

First Advisor

Dr. Khaled EI-Tarabily

Second Advisor

Dr. Mohamed Badawi

Third Advisor

Dr. Mohamed AI-Deeb

Abstract

Soil-borne fungal plant pathogens cause some of the most widespread and serious plant diseases. Root diseases caused by these pathogens such as wilt, root rot, collar rot, foot rot, seedling pre- and post-emergence damping-off are the foremost of problems which are gaining importance from day to day in the world and for which no direct control measures have been evolved so far. Fusarium spp. is a widespread soil-borne plant pathogen and is the causal agents of wilt disease of many economically important crops. Fusarium spp. is a limiting factor in the plant productivity in most agricultural soils. Wilt disease of tomato is a common problem in almost all field and greenhouse tomatoes. The disease is caused by different species of Fusarium and in the United Arab Emirates, Fusarium oxysporum f.sp. lycopersici is the common causal agent of the disease. The disease is an economic threats to commercial growers especially those who grow tomatoes under greenhouse humid conditions. At present, a combination of cultural practices and fungicide applications are used to control the diseases.

The aim of the present thesis was to search for a modem dual plant protection and plant growth-promoting strategies by the application of beneficial rhizosphere-competent actinomycetes isolated from the UAE soils. In addition, improving the performance of the selected bio-control and plant-growth promoting actinomycetes isolates through the application of seaweed extract as a soil organic amendment was the target of the present thesis.

To achieve this, eighty seven actinomycetes (65 streptomycete and 22 non-streptomycete actinomycetes) were isolated from tomato rhizosphere soil collected from a farm in Al Ain, United Arab Emirates (UAE). All isolates were screened preliminary for their ability to produce cell-wall degrading enzymes (chitinase) using colloidal chitin agar. Of the 87 actinomycete isolates, only 31 isolates (25 streptomycete and 6 non-streptomycete actinomycetes) produced large clear zones (> 40 mm) on colloidal chitin agar. The remainder did not produce or produced small clear zones (< 40 mm) and were not included in subsequent studies.

Of the 31 highly active chitinase-producing 31 isolates which produced high levels of chitinase, the most inhibitory 19 isolates (15 streptomycete and 4 non-streptomycete actinomycetes) were examined in vitro for their ability to suppress the growth of F. oxysporum f.sp. lycopersici, a soil-borne fungal plant pathogen causing wilt disease of tomato (Lycopersicon esculentum Mill.) in the UAE. These 19 isolates gave a strong inhibition of F. oxysporum f.sp. lycopersici on colloidal chitin agar.

On highly selective Hussein's fish-meal extract agar medium, used for the detection of antifungal metabolites, only 15 isolates (13 streptomycete and 2 non-streptomycete actinomycetes) out of the 19 tested isolates produced diffusible inhibitory compounds active against F. oxysporum f.sp. lycopersici and were chosen for further in vitro and in vivo studies. The growth of the pathogen was retarded by the diffused metabolites which were produced by the antagonistic actinomycetes.

These 15 promising isolates which produced chitinase and antifungal metabolites active against F. oxysporum f.sp. lycopersici were screened also for their competence as root colonizers and for their abilities to colonize the roots and the rhizosphere of tomato in vitro and in planta. Eight isolates out of the 15 antagonistic isolates completely failed to colonize tomato roots in the root colonization plate assay in vitro 8 days after radicle emergence and subsequently were not included in further studies. The remaining seven isolates were further tested in soil (rhizosphere competence assay) to test their abilities to colonize roots in planta.

Root-colonization abilities of the seven isolates tested showed that roots, and soil particles attached to roots of 21-day-old tomato seedlings were colonized to different degrees by these isolates. Population densities also showed that the isolates were found in the rhizosphere at all depths of the roots, but population densities were significantly (P<0.05) greater in the first 6 cm of the root system compared to other root depths.

Only four isolates (Streptomyces isolates # 8, 11, 42, and 59) which completely colonized the roots up to 14 cm depth in the rhizosphere competence assay were chosen for all further experiments described below. Colonization frequency of the root segments and the rhizosphere soil was greater in plants treated with Streptomyces isolate # 42 and Streptomyces isolate # 59 followed by Streptomyces isolate # 11 and then Streptomyces isolate # 8. Scanning electron microscopy studies of 8-days-old tomato roots colonized with the four Streptomyces isolates showed the presence of extensive mycelia and chains of spores around root and root hairs.

The four most inhibitory Streptomyces isolates (Streptomyces roseodiastaticus isolate # 8), (Streptomyces erumpens isolate # 11), (Streptomyces aurantiacus isolate # 42), and (Streptomyces rameus isolate # 59) produced high levels of chitinase and β-1,3-glucanase in vitro. When the pathogen was presented as the sole carbon source, all four isolates caused extensive plasmolysis, and cell wall lysis of F. oxysporum f.sp. lycopersici hyphae.

The crude culture filtrate of the four antagonistic isolates exhibited antifungal activity and significantly reduced (P<0.05) spore germination and germ-tube growth of the pathogen. The same crude enzyme preparations of the four antagonistic isolates also significantly inhibited (P<0.05) the growth of F. oxysporum f.sp. lycopersici when incorporated into Potato Dextrose agar (PDA).

The most promising four isolates were further examined for their abilities to produce volatile antibiotics, siderophores, indole-3-acetic acid, polyamines, and to solubilize insoluble rock phosphate. In addition to the abilities of the four Streptomyces isolates to produce chitinase, β-1,3-glucanase, diffusible inhibitory antifungal metabolites, and to lyse the hyphae of F. oxysporum f.sp. lycopersici, they varied in their abilities to produce volatile antibiotics, siderophores, indole-3-acetic acid, polyamines, and to solubilize insoluble rock phosphate.

Streptomyces roseodiastaticus (isolate # 8) produced only polyamines. Streptomyces erumpens (isolate # 11), produced volatile antibiotics, indole-3-acetic acid, and solubilized insoluble rock phosphate. Streptomyces aurantiacus (isolate # 42) produced indole-3-acetic acid, polyamines and solubilized insoluble rock phosphate. Streptomyces rameus (isolate # 59) produced volatile antibiotics, indole-3-acetic acid, and solubilized insoluble rock phosphate. None of the four isolates produced siderophores.

These most-promising antagonistic four isolates were subsequently tested in the greenhouse, individually or as a mixture, for their ability to suppress wilt disease of tomato in soil with or without seaweed amendment. The treatment which included all four isolates in soil amended with F. oxysporum f.sp. lycopersici and seaweed was significantly superior to all other treatments in suppressing wilt disease and was nearly as good as the fungicide treatment (Prochloraz application).

The application of the four actinomycetes in the presence of the pathogen either singly or in combination in the absence of seaweed amendments, or in the presence of seaweed amendments, either singly or in combination also significantly (P<0.05) promoted plant growth compared to the controls. In these treatments, there were a significant (P<0.05) increases in the shoot and root lengths and in the dry weights of shoots and roots. The treatment which included all four actinomycetes applied in combination in the presence of seaweed amendments gave the best growth promotion effect in comparison with other treatments.

Microbial activity in soils amended with seaweed extract, were found to be significantly (P < 0.05) higher than in seaweed-non-amended soils, eight weeks after the addition of seaweed extract in the soil indicating the importance of soil amendments on natural microbial activity.

A positive association was evident with their in vitro antagonism and disease reductions in each case. This study clearly showed the potential for the application of antagonistic rhizosphere-competent actinomycetes mixture in a seaweed amended soil, for the management of tomato disease caused by F. oxysporum f.sp.lycopersici in the UAE.

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