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Evaluation of Streptomyces strains for Biological Control of Charcoal Stem Rot of Melon Caused by Macrophomina phaseolina



H.R. Etebarian
 
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ABSTRACT

Biological control of causal pathogen (Macrophomina phaseolina) was investigated by four strains of Streptomyces. Dual culture and cellophane overlay technique were used in vitro assay. All antagonist - host combination were carried out in 4 replicates. Colony area was recorded daily, compared with controls and percentage of growth inhibition was calculated. Glasshouse studies were performed to test the ability of Streptomyces strains incorporated to the soil as a rate of 10g kg-1 potting mix. In other experiment seeds of melon were soaked in Streptomyces suspension and planted in infected soil with pathogen. Streptomyces strains significantly inhibited mycelial growth of Macrophomina phaseolina in dual culture. Streptomyces strains, STL, A20, and A15 inhibited the growth of pathogen by 88.16 to 89.3% and mycelial growth of M. phseolina was reduced 69.99% by Streptomyces strain A22. Cell free metabolites produced by 4 strains of Streptomyces reduced colony area by 80.14-99.66%. The results of glasshouse experiment indicated that when soil of pots inoculated with pathogen + Streptomyces or Streptomyces alone, percentage of healthy plants were significantly greater than in those of pathogen control (p<0.05). Similar results were obtained in seed treatment test.

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H.R. Etebarian , 2006. Evaluation of Streptomyces strains for Biological Control of Charcoal Stem Rot of Melon Caused by Macrophomina phaseolina . Plant Pathology Journal, 5: 83-87.

DOI: 10.3923/ppj.2006.83.87

URL: https://scialert.net/abstract/?doi=ppj.2006.83.87

INTRODUCTION

Charcoal stem rot, caused by the soilborne fungus Macrophomina phaseolina, is a serious disease of many crops associated with drought stress[1]. This disease also is an important disease of melon in Iran and has been reported from different areas of Iran[2]. Charcoal rot affects all cucurbits. Brown leaves of infected plants turn yellow and whiter. The vines may wilt and die, depending on the extent of infection. A green water soaked lesion forms on the stem near the ground level and may produce amber gumming. The lesion may extend 5-15 cm up the vine and as it dries the color changes to tan. Eventually, small black microsclerotia (and sometime pycnidia) form within the lesion giving it a dusty, charcoal appearance. There are a few effective control measure for this disease which include maintaining optimal soil moisture to avoid plant stress, rotation of cucurbits with a small grain crop. Some newly released hybrids show a high level of vine decline. Fumigation has shown some success in the controlling charcoal rot caused by Macrophomina phaseolina[3,4]. Cucurbit should be well supplied with N, P and K and especially with the minor elements and maintain a well-balanced soil fertility to encourage vigorous growth[1,5]. Biological agents could be an important component in the control of M. phaseolina if effective and reliable formulations were readily available and could be integrated with chemical fungicides. Khalifa and Lindel[6] reported that Trichoderma harzianum reduces disease severity of Macrophomina root rot of Melon in Egypt. Streptomyces species also warrant investigation as potential biological control agents for charcoal stem rot, as they have been reported to suppress a number of disease including pink rot of potato caused by Phytophthora erythroseptica[7] , root rot of Bakansia grandis caused by Phytophthora cinammomi[8], damping off of alfalfa seedlings by Phoma medicaginis[9]. Toussaint et al.[10] showed that 11 strains of Streptomyces species protected raspberry plants from disease caused by P. fragariae var rubi. Actinomycetes isolated from soils collected from capsicum crops in Korea have been shown to produce antibiotics active against P. capsici[11]. Faroughi et al[12]. tested 13 strains of Streptomyces for biocontrol of cantaloup root rot caused by Phytophthora drechsleri, they concluded seed treatment or adding of the mixture of 13 Streptomyces strains to infected soil with pathogen increased seedling survival by 43-50%. However, there is little or no information on the efficacy of Steptomyces against charcoal stem rot of melon. The aim of this investigation was to determine the potential of some strains of Streptomyces for biological control of Macrophomina phaseolina through in vitro and glasshouse experiments.

MATERIALS AND METHODS

Pathogen and antagonist strains: The isolate of Macrophomina phaseolina was isolated from diseased plants of melon from the Garmsar area of Iran and maintained on Potato Dextrose Agar (PDA) in the dark at 4°C.

Streptomyces strains A22, A20, A15 and STL were obtained from in ground glasshouse crops of Capsicum near Virginia, South Australia[13]. The strains inhibited the growth of Sclerotium rolfsii Sacc and Phtyophthora erythroseptica in vitro and potting mix[7,13]. Streptomyces strains were maintained on 1/5 M32 agar (1/5 strength of M32 agar medium )[14] in the dark at 4°C .

In vitro inhibition assay: Dual culture[15] and cellophane overlay[16] techniques were used to examine the effects of Streptomyces strains A15, A20 , A22 and STL on mycelial growth of Macrophomina phaseolina. Culture were grown on 15 ml of casein glycerol medium(CGM) agar medium[7,17] in 9 cm petri plates. For dual culture, half of the agar surface was smeared with a suspension of one Streptomyces strain in sterile distilled water (SDW) using a sterile cotton bud. After incubation at 7 days, a plug (5 mm diameter), cut from the leading edge of 4 day-old culture of M. phaseolina on CGM agar medium was placed on the other half of the plate. For controls, CGM agar was inoculated with the pathogen alone. Plates were incubated at 25°C in the dark for 7 days. The surface area of the colonies M. phaseolina was recorded daily, compared with the controls and the percentage of growth inhibition was calculated.

For the cellophane overlay technique, cellophane membrane (Australia Cellophane, Victoria), 9 cm in diameter were boiled in distilled water interleaved with filter paper and autoclaved. A cellophane membrane was placed on the agar in each petri plate and dried in a lamina flow cabinet for 15 min. A 7 day -old culture of one Streptomyces strain, suspended in SDW, was smeared the entire surface of the cellophane. For controls SDW was applied. The plates were incubated in the dark at 25°C for 7 days, after which the cellophane membrane with adhering Streptomyces culture was removed. A plug(5 mm diameter) of M. phaseolina was placed on the culture of the plate previously occupied by the antagonist. The plates were then incubated in the dark at 25°C for 7 days. The surface area of M. phaseolina colonies was recorded daily, compared with the controls and the percentage of growth inhibition was calculated. The data for percentage inhibition of growth at 1, 2, 3 and 7 days in dual culture and the data for 4 and 7 days after inoculation of the pathogen in cellophane technique were pooled and analyzed statistically.

Glasshouse assay for biological control of M. phaseolina on melon
Soil treatment: The ability of Streptomyces isolates to reduce incidence of charcoal and stem rot of melon in glasshouse was investigated. M. phaseolona isolate was grown on potato dextrose agar (PDA) and when they were growing rapidly (in about 10 days) pieces of culture 5x2 cm in size were transferred to 125 mL Erlenmeyer flasks containing autoclaved sand -corn meal medium (110 g sand, 6 g corn meal, 20 mL sterilized water). The flasks incubated at 25°C for 30 days. Containing of each flask were mixed with 2500 g of autoclaved potting mix and placed in 20 cm pots.

Inoculum of Streptomyces isolates was prepared as follows: wheat barn were soaked for 1 h and then transferred to 125 Erlenmeyer flask and autoclaved for 1 h at 121°C on two successive days. Isolates A13, A15, A20 and STL were grown separately on CGM at 25°C for 7 days. And ¼ of the contents of one petri plate were added to each flask, mixed with wheat barn and incubated at 25°C for 21 days. Wheat barn infected with the Streptomyces, were combined and blended in SDW to make slurry. The rate of inoculum which were applied to potting mix, was 10 g infected barn kg–1. Treatments comprised: Streptomyces alone, mixed Streptomyces isolates + M. phaseolina and control without antagonist and pathogen. Seeds of Garmsar native melon cultivar were surface disinfected by soaking in 0.5% sodium hypochlorite for 3 min then rinsed three times in SDW. Fifteen seeds were sown in each pot. There were four replicate pots per treatment arranged in a completely randomized design. Plants were maintained in the glasshouse without supplementary lighting from April to June(spring) in Pakdasht Tehran. Pots were watered at 2 or 3 days intervals until emerge and daily thereafter.

Seed treatment test: The methods and materials for preparation of M. phaseolina inoculum were the same as described for soil treatment test. Inoculum of Streptomyces isolates were prepared as follows:

Isolates of Streptomyces A13, A15, A20 and STL were grown separately on 9 cm petri plate containing CGM medium at 25°C for 7 days. Bacteria were harvested from surface of plate and washed with 10 mL of SDW and suspended in 0.05% tween 20. Suspension of 4 isolates of Streptomyces were combined equally . The melon seeds were disinfected as mentioned above and soaked for 30 min in Streptomyces suspension and air died in laminar-air flow hood. For adhearing of bacteria to seed surface methyl cellulose was used[18]. The other methods and materials were the same as soil treatment test. In both experiments the percentage of healthy plants were determined 40 days after planting.

Statistical analysis: Data on percentage inhibition of growth and percentage of healthy plants were subjected to arsin square root transformation, before analysis, Analysis of variance was performed and means were separated using Duncan’s Multiple Range Test at p<0.05[19].

RESULTS AND DISCUSSION

Effect of Streptomyces isolates on mycelial growth of M. phaseolina in vitro: All isolates of Streptomyces inhibited mycelial growth of M. phaseolina in dual culture. Mycelial growth was reduced by 14.86-36.00 and 89.12-98.79 by Streptomyces isolates, 1 and 7 days after inoculation, respectively (Table 1)

Cell free metabolites produced by Streptomyces isolates reduced colony area of M. phaseolina by 80.14-99.50 and 83.56-99.66, 4 and 7 days after inoculation, respectively (Table 2).

Table 1: Percentage of growth inhibition of M. phaseolina by different strains of Streptomyces in different times after inoculation (dual culture)
Image for - Evaluation of Streptomyces strains for Biological Control of Charcoal Stem Rot of Melon Caused by Macrophomina phaseolina
Data are means of four replicate plates. Numbers within columns followed by a common letter are not significantly different at p<0.05, according to Duncan,s Multiple Range Test. Data were subjected to arcsin square root transformation before analysis. Data are expressed as control without antagonists.

Table 2: Percentage of growth inhibition of M. phaseolina by different strains of Streptomyces (cellophane overlay Technique)
Image for - Evaluation of Streptomyces strains for Biological Control of Charcoal Stem Rot of Melon Caused by Macrophomina phaseolina
Data are means of four replicate plates. Numbers within columns followed by a common letter are not significantly different at p<0.05, according to Duncan,s Multiple Range Test. Data were subjected to arcsin square root transformation before analysis. Data are expressed as control without antagonists.

Table 3: Antagonistic effect of combined Streptomyces strains on the disease incidence caused by M. phaseolina
Image for - Evaluation of Streptomyces strains for Biological Control of Charcoal Stem Rot of Melon Caused by Macrophomina phaseolina
Data are means of four replicate pots. Numbers within columns followed by a common letter are not significantly different at p<0.05, according to Duncan,s Multiple Range Test. Data were subjected to arcsin square root transformation before analysis. Data are expressed as control without antagonist and pathogen.

Image for - Evaluation of Streptomyces strains for Biological Control of Charcoal Stem Rot of Melon Caused by Macrophomina phaseolina
Fig. 1: Effect of seed treatment of Streptomyces on disease incidence of charcoal stem rot (left; Macrophomina phaseolina + Streptomyces: right; Macrophomina phaseolina only).

Image for - Evaluation of Streptomyces strains for Biological Control of Charcoal Stem Rot of Melon Caused by Macrophomina phaseolina
Fig. 2: Effect of soil treatment of Streptomyces on disease incidence of charcoal stem rot (left; Macrophomina phaseolina + Streptonyces: right; Macrophomina phaseolina only).

Biological control of Streptomyces on melon in glasshouse conditions: Survival of seedling in pots treated with M. phseolina plus Streptomyces was similar to that in plants inoculated with Streptomyces only or control without antagonist and pathogen. Percentage of healthy plants in treatment Streptomyces + M. phaseolina were significantly greater than those M. phaseolina only in both seed and soil treatment tests (Table 3, Fig. 1 and 2 ). All strains of Streptomyces, inhibited growth of M. phaseolina in vitro, using dual culture and cellophane overlay techniques. The presence and size of the zone of inhibition suggested that this effect was due to the production of antibiotics by Streptomyces[7,9,20-22].

The mode of action of Streptomyces appeared to be antagonism by the production of Tubercidin, produced by Streptomyces tubercidicus and S. violaceoniger against Phytophthora capsicii[23] , Geldamycin produced by S. hygropicus against Rhizoctonia solani[24].

The isolates tested here have also potential against other soil borne pathogens, Cell free metabolites of Streptomyces strains tested inhibited growth of Phytophthora erythroseptica the causal agent of pink rot of potato in vitro. The lesion size in Pontiac potato treated with P. erythroseptica and combined Stretomyces isolates (mean of 13.25 mm) were significantly less than of that pathogen alone (mean of 54.75 mm). The yield of tubers from Pontiac plants treated with combined Streptomyces and P. erythroseptica (mean 16.5 g fresh weight potato) were significantly greater than in control inoculated with the pathogen alone (mean 6.77 g/pot)[7].

The dry weight of shoots and roots of melon were not determined in this investigation, but in the absence of the pathogen, some actinomycetes isolates significantly increased shoot and dry weight. Such increases have been reported in cauliflower[25], wheat[26] and also increase of potato yield have been reported[7]. This phenomenon may be related to the ability of actinomycetes to produce growth regulator[27,28].

The use of actinomycetes to control fungal pathogens has advantages as they are not affected by fungicides and therefore, can be used as a component of integrated disease control including chemical control.

In this study percentages of healthy plants in treatments Streptomyces + M. phseolina were 100 and 96.65 in seed and soil treatment test, respectively. Seed treatment with Streptomyces is more acceptable than soil treatment because it is easier and low cost.

The effect of age of inoculum and means of application on the ability of Streptomyces to give consistent control charcoal stem root rot should be investigated. Similarly, the timing of the application of antagonists and the pathogen should be studied. In commercial crops, M. phaseolina are also present as sclerotia in infested soil prior to planting, however, the effect of Streptomyces on sclerotia has not been determined. In this study, the antagonists were applied , at the time of planting, to soil which had been infected one day previously and it would be of value to examine the effects of infecting the soil both earlier than, and the time of planting. A biological control product which was effective when applied at the time of planting would be more likely to be accepted by growers than one which required additional cultivation. This means of application would also reduce the need for long-term survival of the antagonists in the soil, which may be limiting factor in the biological control of soil- borne fungal pathogens[29,30]. In conclusion, the Streptomyces strains tested here reduced disease severity in melon plant seedling in the glasshouse. Future research will involve studies of the mechanism involve. These isolates warrants further investigation for their ability to control of charcoal stem root rot especially in commercial situation. An integrated approach using a combination of Streptomyces and Trichoderma species such T. harzianum T39 and T. virens DAR 7420 (Etebarian unpublished) may allow reduction of disease incidence in different climate and different soils.

ACKNOWLEDGMENTS

The financial support of vice chancellor for research, University of Tehran is gratefully acknowledged. The technical assistance of Mehrnosh Mohammadi Fard and Asghar Zarei Sarabi are appreciated. The author also thanks to Dr. E.S. Scott , Department of Applied and Molecular Ecology, The University of Adelaide for supplying of Streptomyces cultures.

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