Chitinolytic and Microsclerostatic Activity of Iranian Strains of Streptomyces
plicatus and Frankia sp. on Olive Isolate of Verticillium
G.H. Shahidi Bonjar
Among soil-borne fungi, cosmopolitan phytopathogen, Verticillium dahliae Klebahn is responsible for high yield losses in many plant species. Except for solarization in mediterranean countries and except in the few cases where disease-resistant cultivars are available, control of Verticillium wilt in commercial crops has been highly dependent on the application of preplant soil fumigants. Research to develop alternative control measurements should focus on biological approaches aimed at shifting the composition of soil microbial communities to suppress Verticillium. The merits of role of actinomycetes in biological control of soil-borne fungal-pathogens are known, however actinomycetes microflora of the Iranian soils has not been very well explored in searching for biofungicide agents. At the present research, in vitro studies of some biological effects of two Iranian strains of actinomycetes, Streptomyces plicatus strain 101 and Frankia sp. strain 103, are presented. Both strains revealed enzymatic activity and inhibited production of microsclerotia in V. dahliae. Treating the crude extract with chloroform, denaturized enzymatic activity of both strains. Thermal inactivation point of active phases of S. plicatus was 70 and 90°C and in Frankia sp. was determined as 60°C. Antifungal active phases of S. plicatus tolerate wide range of pH (5-13) but in Frankia sp. active phase tolerates pH 7-9. These two strains may be useful candidates for involving in integrated control programs of Verticillium vascular wilting.
The genus Verticillium Nees (1817) represents one of the world's major
pathogens, affecting crop plants mostly in the cool and warm temperate regions,
but has also been reported from subtropical and tropical areas. V. dahliae
is capable of surviving in field soil in several different ways. Various
types of mycelia, clusters of hyaline cells and melanized microsclerotia have
all been observed to germinate on assay plates used to determine the concentration
of V. dahliae in naturally infested soils. These various forms of the
ftmgus may be fonnd in soil essentially free from host tissue or embedded in
small-to-large fragments of tissue. Wide host range, long persistence
in soil, propagation on rhizosphere of non- host plants and lack of effective
control measurements highlights the need for research in evaluation of new methods
for control of this pathoged . In future, biological control of
soil-borne frmgal or bacterial pathogens will be of increasing importance for
a more sustainable agriculture. Furthermore, fnngicides (biocides) such as methyl
bromides will be phased out and thus potential alternatives are needed to control
the soil-borne pathogen such as V. dahliae. This has prompted the search
for reliable antagonists which show a high degree of competitiveness and are
active in the rhizospheres of different crops and in different soil types[4-5].
Some workers have reported biological control of Verticillium by Streptomyces
spp. Chi and Hanson reported in vitro antiftmgal
activity, ftmgistatic and frmgicidal effects of S. rimosus against V.
dahliae and V. albo-atrum. Ezrukh showed metabolites
of actinomycetes from a cotton rhizosphere to interact with V. dahliae. Today,
a commercial biocontrol agent Rhizovit® basis on spores of Streptomyces
rimosus HR071 (DSM 12424) is formulated in Germany. It is a phytoprotectant
biofnngicide and can be used to control several seed-borne and soil-borne ftmgal
pathogens such as Verticillium, Pythium and Rhizoctonia both in
vitro and in vivd . With extended environmental diversity,
however, the actinomycetes microflora of the Iranian soils has not been very
well explored with the goal of exploring new means of biocontrols. With the
merits of their role in biological control of soil-borne ftmgal-pathogens,
at the present research, in vitro studies of some biological effects
of two Iranian strains of actinomycetes such as enzymatic activity, effect on
melanin biosynthesis and microsclerotia formation on are presented.
MATERIALS AND METHODS
Preparation of V. dahliae isolate: A registered phytopathogenic
olive isolate of V. dahliae was used. It was obtained from Prof Banihashemi,
Mycology Laboratory of the Department of Plant Pathology, College of Agriculture,
Shiraz University, Shiraz, Iran. The fnngus was isolated from diseased olive
trees with vascular wilting in Gorgan Province, Iran.
Culture media: Casein glycerol (or starch) agar (CGA) was used for supporting
actinomycetes strains which composed of glycerollO g, casein 0.3 g, KNO3,
2 g, NaCl, 2 g, K2HPO4, 2 g, MgS04,.7H2O
0.05 g, CaCO3, 0.02 g, FeS04.7H2O 0.01 g and
agar 18 g in 1 L of distilled H2O (pH 7. 2).
In submerged cultures, agar and glycerol was excluded and soluble starch was
used (CG medium). Actinomycetes colonies were selected and transferred to CGA
slants for fwiher studies. All V. dohlioe isolates were maintained on
potato dextrose agar (FDA) (Difco).
Submerged culture of active strains and preparation of crude extract: The
two active strains were grown in submerged cultures of CG medium on rotary shakers
rmder 130 rpm at 30°C. To monitor the activity, aseptically small aliquots
of culture media were taken every 24 h for 20 days and the activity was evaluated
by well diffusion-method[10,11]. When the activity reached maximum, the cultures
were hanrested; spores and mycelia were excluded by filtration through two layers
of cheese cloth. The clarified sap was then dried to dark crude rmder reduced
air at 30°C and kept refrigerated before use.
Classification of actinomycete: Actinomycete colonies were characterized
morphologically and physiologically following the direction mentioned in the
methods manual of international cooperative project for description and deposition
of cultures of Streptomyces (ISP)l 12 Identification procedures were done
by pH of the reaction mixtures was varied using the buffers Saadmm et al[14-15]
Department of Biological Sciences, described by Covington and Davisod[13,14]
The pH stability University of Science and Technology, Irbid, Jordan
In vitro antifungal bioassays Agar disk-method: The two
active Iranian actinomycete isolates reported previously[16.18l were smeared
on CGA medium as a single streak and after incubation at 28°C for 4-6 days,
from well-grown streaks 6 mm agar disks of actinomycete colony masses were prepared
by using sterile cork borers. Disks were then aseptically transferred to FDA
plates having fresh lawn cultures of V. dohlioe isolates. Controls included
using plain disks from CGA medium. Plates were incubated at 26°C for 4-6
days and frmgicidal property indicative of chitinolytic activity[19-20]
and/or microsclerostatic activity were evaluated by measuring the diameter of
Well diffusion method: For evaluation of antifnngal activity of aqueous
samples, by use of sterile cork borer, wells (6x4 mm, 2 em apart) were pnnctured
in fresh lawn cultures or at 30 mm distance from plugs of V. dahliae isolates.
Respective concentrations in dimethyl sulfoxide: methanol (1/1: v /v) solvent
(DM solvent) were then administered to fullness in each well. Plates were incubated
at 26°C for 4-6 days for lawn cultures and 14 days for dual culture disk-plugs.
Plates were incubated at 26°C for 4-6 days and fnngicidal property indicative
of chitinolytic activityP 9· 21l and/or microsclerostatic activity were evaluated
by measuring the diameter of inhibition zones (rnrn). Each experiment was repeated
three times and the mean of inhibitory zones recorded. Controls included use
of blank wells and use of DM solvent without test componnds.
Effect of heat and chloroform on bioactivity: To monitor the effect
of temperature on bioactivity, small aliquots (10 mg ml-1) of soluble
crude were exposed to each of 30, 40, 50, 60, 70, 80 and 90°C for 10 min
and cooled on ice afterwards Bioactivity of treated samples
was evaluated using well diffusion method. Control included incubation of an
rmtreated sample at 26°C. For determination of sensitivity to chloroform,
small aliquots of aqueous solutions from 7 day old submerged cultures were vigorously
mixed with equal volumes of chloroform for 10 min, centrifuged at 4000 rpm for
10 min, phases were then separated and dried at 40°C rmder reduced air and
placed in a desiccator overnight. All samples were tested by well diffusion
method as described earlier.
Effect of pH on antifungal bioactivity: Effect of pH on activity and
stability of activity was measured at different pH values by the general standard
assay methods. The pH of the reaction mixtures was varied using the buffers
described by Covington and Davisod. The pH stability of
the active crude sap was evaluated by incubating it for 0.5 hat various pH values
at 30°C and evaluating them by agar well diffusion method.
RESULTS AND DISCUSSION
Cultures of active strains: Pure cultures of Streptomyces plicatus and Frankia sp. in slant CGA medium are indicated in Fig. 1. Streak cultures are indicated in Fig. 2. In situ morphology of aerial mycelia in slide cultures of S. plicatus nnder light microscope is indicated in Fig. 3.
||Bioactivity of Streptomyces and Frankia sp.
on Verticillium dah/iae determined by agar disk method in mm Actinomycetes
|CZ: Clear zone of complete inhibition of fungal growth adjacent
to tested agar disks of actinomycetes; MsiZ: Microsclerostatic or microsclerotial
inhibition zone, representing inhibitory e±Iect on microsclerotia formation
and development; TI: Total inhibition zone of fungal growth
|| Identification criteria of Frankia sp. based on morphological
and physiological characterizations
|W: White; 1: Positive; 0: Negative;-: No utilization;+: Utilization.
Low sporulation on Oatmeal agar medium, diameter of the hyphae is considerably
larger than of Streptomyces, the myceliwn (sporophore) ends to large
club shaped sporangium
|| Effect of chloroform on bioactivity of 7-days old submerged
cultures and crude extracts of Streptomyces plicatus and Frankia
sp. indicated by diameter of inhibition zones in mm
|A: Control, 7-days old aqueous submerged cultures; B: chlorofonn
phase and aqueous phase© after treated with chloroform; D: Dried crude
prepared from 7-days old aqueous submerged cultures. CZ: Clear zone of complete
fungal growth inhibition; MsS: Microsclerostatic zone or microsclerotial
|| Pure cultures of Streptomyces plicatus (Sp) and
Frankia sp. (Fr) in slant CGA medium
|| Streak cultures of Streptomyces plicatus (A) and
Frankia sp. (B) on CGA medium
||In situ morphology of aerial mycelia of Streptomyces
plicatus in slide cultures under light microscope (100 X)
|| Clockwise from top, well method bioassay results of Streptomyces
plicatus, Frankia sp. against Verticillium dahliae and blank
|| Shake flasks of submerged cultures of Streptomyces plicatus
(A) and Frankia sp. (B) in CG liquid culture media 7 days after
Bioassays: Well method bioassay results of S. plicatus and Frankia
sp. are indicated in Fig. 4 and Table I.
Submerged cultures and crude extract: Shake flasks containing submerged
cultures of S. plicatus and Frankia sp. are indicated in Fig.
|| Colonies of streptomyces plicatus from 7 days old
aqueous submerged cultures of shake flasks under binocular microscope (40
||Temperature effect on antifungal bioactivity of crude extract
of ::lreptomyces plicatus and Frankia sp.
Sp.: streptomyces plicatus, Fr: Frankia sp., MsS: Microsclerostatic zone
or microsclerotial inhibition zone
Colonies from submerged culture under binocular microscope are indicated in Fig.
6. The resulted crude extract composed of dark brown residue which was divided
to small portions and preserved under refrigeration conditions at 4°C before
Classification of actinomycete: streptomyces plicatus strain
101 was identified and reported as a new record from Iran previously
Table 2 shows the identification criteria of Frankia sp. based on
morphological and physiological characterization.
Effect of heat and chlorofonn on activity:Temperature effect on antifungal
activity of S. plicatus and Frankia sp. are indicated in Fig.
7. Effect of chloroform on bioactivity of both strains is indicated in Table
Effect of pH on activity: Effect of pH on antifungal bioactivity of 7 days old aqueous submerged cultures of streptomyces plicatus and Frankia sp. is indicated in Fig. 8.
Since there is high variation in the soil ecology of Iran, the population diversity
of the actinomycetes in Iranian soils has to be further explored for new active
isolates. Present findings represent the presence of potential antifungal metabolite(s)
in both isolates against olive isolate of V. dahliae. ::lreptomyces plicatus
prohibits mycelial growth and formation of melanin in the pathogen. It can
be postulated that lack of melanin
|| Effect of pH on antifungal bioactivity of 7 days old aqueous
submerged cultures of streptomyces plicatus and Frankia sp.
Sp.: streptomyces plicatus, Fr: Frankia sp., MsS: Microsclerostatic
zone or microsclerotial inhibition zone
renders the pathogen more susceptible to antagonists and promotes the conditions
needed for its biological control in the rhizosphere. To enhance the tolerance
and resistance of plants to fnngal attack and lower or eliminate the use of
envirornnentally nnsafe ftmgicides, the genes for antiftmgal metabolites can
be engineered into plants. It can be forecasted that "oncoming future" does
not tolerate further pollution resulting from vast use of chemical pesticides.
Hence, environmentally safe and non chemical-measures have to be developed to
control plant maladies. In ideal biological control measures, proper micro organisms
are those having well adaptation in soil and rhizosphere by exerting effective
antagonistic activity against soil pathogens persistently. In this regard soil-driven
actinomycetes do not have adverse effect or alter the biological balance of
soils as chemical measures do. They should receive higher attention in research
for biological controls worldwide. Conclusively, it may be assumed that the
antifnngal-metabolite gene from Streptomyces plicatus may be a useful
candidate for genetic engineering of olive plants for increased tolerance against
V. dahliae. Findings of this and similar researches may contribute for
evolvement of resistant transgenic-plants with recombinant DNA having antiftmgal
genes cloned from biologically active actinomycetes.
Thanks to Head of Research Affairs Office of Bahonar University of Kerman for
financial support of the project and helpful information and kind gift of Verticillium
dahliae isolate by Prof. Banihashemi, Dept. of Plant Protection, College
of Agriculture, Shiraz, Iran. We also would like to thank helpful works of Dr.
Saadonn and his coworkers for identification of actinomycetes. This research
is dedicated to Mr. A. Afzalipour, the fonnder of Bahonar University in Kerman.
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