Biological control of citrus green mould , Penicillium digitatum , by antifungal activities of Streptomyces isolates from agricultural soils

Streptomyces strains can control postharvest citrus fruit diseases caused by Penicillium digitatum. One hundred and ten (110) Streptomyces strains were isolated from agricultural soils and screened against P. digitatum. The antagonistic activities of the isolates were determined through dual culture technique. In the 110 isolates, only strain 328 showed strong antifungal inhibitory effects. Streptomyces strain 328 showed a maximum biomass value (1.85 g/L) after 72 h incubation in yeast extract, malt extract, glucose (YMG) medium; in starch casein medium, the highest value was 0.68 g/L after 96 h incubation. The metabolites produced in starch casein medium showed the maximum inhibition zone against P. digitatum (28 mm diameter). Streptomyces strain 328 metabolites have molecular mass higher than 2000 and they have fungistatic mode of action against P. digitatum. In in vivo assays, the selected isolate has significantly reduced severity of green mold on crops which have been stored under 24°C for 28 days as compared to the controls. The inhibition effect of about 95% was shown by strain 328.


INTRODUCTION
Postharvest losses of fresh fruits and vegetables may reach very high values depending on species, harvest methods, length of storage and marketing conditions, Postharvest diseases often account for a major part of losses (Brown and Chambers, 1996;Bull et al., 1997;Pailly et al., 2004) and their control requires use of a large amount of fungicides (Wilson et al., 1993;Holmes and Eckert, 1999).Biological control of postharvest diseases by antagonistic microorganisms seems to be a promising alternative to fungicides (Qing and Shiping, 2000;Fan and Tian, 2001;Long et al., 2005;Zhang et al., 2005).Understanding the methods of action of antagonisms is essential to allow the use of antagonists under partial conditions and to enhance their biological control while protecting the human health and the environment.Several modes of action have been documented for the antagonistic activity of biological control agents: they act by multiplying on the fruit surface or within wounds on the fruit, then by competing for space and nutrient at an infection court on the *Corresponding author.E-mail: shahdaei_s@yahoo.com.Tel: 00989131406552.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License commodity, by antibiosis, by restricting the action of hydrolytic enzymes produced by the pathogen, by producing enzyme to degrade pathogen cell walls, and/or by direct parasitism of the pathogen (Jijakli and Lepoivre, 1998;Bar-Shimon et al., 2004;Long et al., 2005).Green mould, caused by Penicillium digitatum Sacc., is among the most economically important postharvest diseases of citrus worldwide.It has been estimated that losses of citrus fruit due to green mould infection is 30-50% in China (Boxun et al., 2002), the world's largest producer of citrus fruit.One major research focus in this area has been the selection and development of effective and environment-friendly agents for biological control of this disease.The purpose of these experiments was to evaluate the commercial potential of Streptomycetes for the control of postharvest decay of citrus fruit.At present, we have targeted our efforts toward the control of green mould.
Streptomyces spp.are one of the most attractive sources of biologically active substances such as vitamins, alkaloids, plant growth factors, enzymes and enzyme inhibitors (Omura, 1986;Shahidi, 2003).Soil Streptomycetes are one of the major contributors to the biological buffering of soils and have roles in decomposition of organic matter conductive to crop production (Gottlieb, 1973;Kieser et al., 2000).For this research, 110 isolates of Streptomycetes were isolated from agricultural soils of Kerman of Iran and screened against P. digitatum in vitro and in vivo.The objective of this study is to isolate Streptomyces strains having antagonistic properties with the aim that they can serve as gene donors in developing resistant transgenic plants and used for soil amendments as biofertilizer or biofungicide in biological control of the tested pathogen.From all tested isolates of Streptomycetes, 11 Streptomyces strains showed high in vitro anti green-mold activity.

Culture media
P. digitatum was obtained from Iran Plant Protection Research Institute (IPPI).The pathogen was cultured on potato dextrose agar (PDA).Spores were harvested by flooding the surface of 10 daysold cultures with distilled water; the inoculum concentration used was 10 6 spores/ml.This concentration is commonly used in citrus postharvest experiments (Eckert and Brown, 1986).Casein glycerol agar (CGA) was prepared from basic ingredients as described by Küster and Williams (1964) and used as Streptomycetes culture.

Soil sampling and isolation of Streptomycetes
Samples of soil were collected from grassland, orchards and vegetable fields in different parts of Kerman, Iran (Kerman is one of the major region that produces varied agricultural crop, particularly citrus).Several samples were selected using an open-end soil borer (20 cm in depth, 2.5 cm in diameter) as described by Lee and Hwang (2002).Soil samples were taken aseptically from a depth of 10-20 cm below the soil surface.Samples were air-dried at room temperature for 7 to 10 days and then passed through a 0.8 mm mesh sieve and were preserved in polyethylene bags at room temperature.Ten gram samples were mixed with sterile distilled water (100 ml) and the mixtures were shaken vigorously for one hour and then allowed to settle.One milliliter of soil suspensions (diluted 10 -1 ) were transferred to 9 ml of sterile distilled water and subsequently diluted to 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 .Inoculum consisted of adding aliquots of 10 -3 to 10 -6 soil dilutions to autoclaved CGA (1, 25 ml -1 CGA) at 50°C and then poured into plates.Three independent replicates were considered for each dilution.Plates were incubated at 30°C for up to 20 days.The Streptomyces colonies were isolated and incubated at 28°C for one week and stored in a refrigerator as pure cultures (growing a single clone on medium is indicative of pure culture) and at the end 110 Streptomyces isolates were screened.

In vitro studies
To estimate the antifungal activity of the Streptomyces isolates against pathogen, bioassays were done using agar disk method as described by Shahidi Bonjar (2003) and Aghighi et al. (2004) and the ratings used were modified by Lee and Hwang (2002) and El-Tarabily (2000).Antifungal activity was defined as growth inhibition against pathogenic fungi and was calculated by subtracting the radius distance (mm) of fungal growth obtained in the control and antagonist plates.Reference values to evaluate inhibition were: slight (5 to 9 mm), moderate (10 -19 mm) and strong (> 20 mm).

Growth kinetics and metabolites production for Streptomyces strain 328
Kinetics studies were carried out with starch casein and YMG media (g/l : yeast extract, 4; malt extract,10; glucose, 4; K2HPO4 , 2; casaminoacids, 0.1%; pH 7) to optimize Streptomyces strain 328 growth and metabolites production.50 ml of YMG and starch casein media were inoculated with 5 ml of a cell suspension (1.5 10 8 cel/ml) of Streptomyces strain 328, incubated for 7 days at 28°C, samples were taken every 24 h.Biomass was separated by filtration and was then dried.The supernatant was used to carry out antagonism assays in solid medium and in vivo (Maldonado et al., 2010).

Determination of minimum inhibitory concentrations (MIC)
To measure the MIC values, two-fold serial dilutions of 50, 25, 12.5, 6.25, 3.125, 1.562, 0.781, 0.390 and 0.187 mg ml -1 of the crude extract were prepared in DM solvent and assayed by well diffusionmethod as described by Shahidi (2004).The MIC was defined as the lowest concentration able to inhibit any visible fungal growth.All data represent average of three replicated experiments.

Polarity detection rate of active crude extract in organic solvents
To evaluate the relative polarity of the active principle(s) present in the crude extract, 2 ml of each of H 2 O, methanol and chloroform were added to 20 mg pulverized-crude samples separately and vortex for 20 min.Each sample was then centrifuged at 3000 rpm for 15 min using a bench low speed centrifuge.Supernatants and pellets were separated at 50°C and assayed at concentration of 20 mg ml -1 by agar diffusion-method (Bonjar, 2004).

Determination of thermal inactivation point (TIP)
Small aliquots (10 mg ml -1 ) of soluble crude extract were exposed to 30, 40, 50, 60, 70, 80 and 90°C for 10 min and cooled on ice afterwards to monitor the effect of temperature on bioactivity.For temperatures over 90°C, heat oil was used.Bioactivity of treated samples was evaluated using well diffusion method.Control included incubation of an untreated sample at 28°C (Nawani and Kapadnis, 2004).

Detection of fungicidal activity and metabolites mode of action
Small blocks of inhibition zones (1 mm 3 ) of Streptomyces strain 328 against P. digitatum was transferred to fresh PDA plates and incubated for seven days at 24-28°C.During incubation, growth or lack of growth of the fungus was investigated both visually and microscopically.Rejuvenation of growth was indicative of fungistatic and lack of growth represented fungicidal properties of the antagonist.

Chloroform assay for detection of antibiotic
Spore suspensions (approximately 10 8 spores ml -1 ) of individual isolates were dotted (10 -6 l per spot) onto 15 ml starch casein agar plates, five dots per plate.Plates were incubated at 28°C for 3 days (Davelos et al., 2004).Dotted isolates were killed by inverting the uncovered Petri plates over 4 ml of chloroform in a Watch glass for 1 h.Watch glasses were removed, and plates were aerated in a fume hood for 30 min to permit evaporation of chloroform.Plates were subsequently overlaid with 15 ml of 1% water agar and inoculated with 10 -6 l of test isolate, P. digitatum, (approximately 10 8 spores ml -1 ) spread uniformly over the surface of the agar.Plates were incubated at 24°C for four to five days.The size of any zone of growth inhibition of the overlaid isolate surrounding any dotted isolate were measured in millimeters from the edge of the dotted colony to the edge of the cleared zone.

Metabolite molecular mass estimation
A benzoylated dialysis tube (SIGMA) capable of separating molecular mass compounds between 1200 and 2000 M.W. was used.Five millilitres of metabolites were dialyzed at 4°C, 6 h in phosphate buffer 0.2 M, pH 7. Recovered metabolites from the dialysis tube were sterilized by filtration and assayed for in vitro antagonism against P. digitatum.

Scanning electronic microscopic
Mycelial morphology of Streptomyces strain 328, was showed by using scanning electronic microscopic (CAM SCAN -MV 2300).Preparation for the scanning electron microscope consisted of using the culture held by a piece of double stick scotch tape.The stubs were coated in a sputter coater for 2 min.Afterwards, the specimens were viewed and digital electron micrograph were prepared at magnification of 6000 to 20000x with an accelerating voltage of 20 kv accordingly.

In vivo antagonism assays
In vivo assays were carried out with Streptomyces strain 328 to study its inhibitory activity against P. digitatum.Fresh orange cultivars Valencia of uniform size and maturity, without wounds, were used in this study.They were picked and washed for 10 min in (5) Oranges treated as in 3 and 4, but with the addition of 20% waxwater emulsion.The fruits were sprayed, allowed to dry, placed in boxes with moistened paper towels to maintain 85% humidity and covered with plastic.They were kept for three weeks at 4°C and 1 week at room temperature to reproduce the storage conditions until reaching their final destination.The results were expressed as percentage of diseased oranges.The results reported are the average of triplicate determinations (Maldonado et al., 2010).

Experimental design and data analysis
The experiments were arranged in a completely randomized design.Each treatment was replicated three times with ten fruits for each replication.The experiments were repeated twice.Extent of disease was planned at each of the four inoculation sites by assessing presence or absence of soft rot symptoms after four day.Statistical analysis was performed with SPSS software package version 15.00 for windows.Analysis of variance and Tukey test of Post Hoc were used for analysis of recorded percentages of mean values.Significant factors in the univariate analysis at first step were entered to multivariate comparisons.

Antifungal bioassays
From tested Streptomyces isolates, eleven isolates were active in dual culture methods, showing two antagonists and the suppressive reaction of P. digitatum.Figure 1 shows bioassay results of Streptomyces strain 328 against P.digitatum measured in agar disk-method.In both methods Streptomyces strain 328 show antifungal inhibitory effects on the pathogen.

Streptomyces strain 328 in in vitro antagonism against several fungal strains
The Streptomyces strain 328 culture and its metabolites against P. digitatum and P. italicum (because P. italicum like P. digitatum produce common mold on citrus) were studied and both could inhibit fungal growth (Figure 2).Table 1 shows that the inhibitory effect was higher in the test carried out with Streptomyces strain 328 culture.

Monitoring antagonistic activity and growth curve
Activity reached maximum after five days in rotary cultures.In shake cultures, this interval was used to harvest cultures to prepare crude extract for use in further investigations.Activity versus post seeding time in rotary

Polarity detection rate of active crude extract in organic solvents
Solubility results are indicated in Table 3.The results show more than one active principle involved since activity is traceable in polar solvents of H 2 O, methanol.

Determination of TIP
Bioactivity of active isolate diminished to zero at 160°C.

Detection of fungicidal activity and metabolites mode of action
P. digitatum was able to grow in PDA medium after their spores were exposed to Streptomyces strain 328 metabolites at 28°C for five days.Based on the result we

Solvent
Fraction Activity assumed that Streptomyces strain 328 metabolites presents fungistatic mode of action.

Chloroform assay
Among 11 effective Streptomyces strain, 3 strains of 328, 263 and 394 retained their antifungal activities after exposure to chloroform.

Molecular mass evaluation for Streptomyces strain 328 metabolites
P. digitatum growth inhibition produced by Streptomyces strain 328 metabolites showed identical levels as experiential without dialysis and after this treatment.We assumed that benzoylated dialysis tube retained Streptomyces strain 328 metabolites and they have a molecular mass higher than 2000.

Scanning electron microscope studies
Scanning electron micrograph of mycelia of Streptomyces strain 328 is indicated in Figure 6 (this picture is necessary for determination of spore level and chain morphology to identification of streptomyces species).

DISCUSSION
In vitro assays of Streptomyces isolate no.328 metabolites presents higher inhibitory effect than Streptomyces culture.This effect could be attributed to competition for space, nutrients, etc.The YMG medium was better than starch casein medium for Streptomyces growth and its metabolites production.These results partially coincide with those obtained by Sabaratnam and Traquair (2002) and Maldonado et al. (2010); the authors also observed that maximum biomass production occurred after three days fermentation in the same conditions.The highest Streptomyces isolate no.328 biomass was 1.85 g/l in YMG medium after 96 h incubation, but grew poorly in starch casein medium, which, Sabaratnam and Traquair (2002) suggested as better than other media.In the case of dual culture technique, only moderate to slight inhibitions were observed.Aghighi et al. (2004) , 2004).Our findings represent the presence of potential antifungal metabolite(s) Streptomyces strain 328 against P. digitatum.Antifungal activity of the isolate found in this study highlights its importance as a candidate for further investigation in biological control of the world-wide destructive citrus postharvest disease.Further works on the project would be focused on field and long-term storage evaluation of the antagonist against the pathogen in the cosmopolitan malady of citrus postharvest disease.The genes encoding many antifungal characteristics are currently being used by agribusiness to create genetically modified plants that have increased fungal resistance in the field or packing house.We believe that the results of these findings can form the avenue for production of resistant transgenic-plants with recombinant DNA having antifungal genes cloned from biologically active Streptomyces isolates which would lead to environmentally safer measures in plant-pest management.

Figure 1 .
Figure 1.Suppressive reaction of two Streptomyces isolates on Penicillium digitatum.up: Streptomyces isolate 328 and down: Streptomyces isolate 223.Center: colony of Penicillium digitatum agar disk which its growth towards the antagonists is inhibited clearly.

Figure
Figure 4. G pH 7 at 28°e

Figure 5 .
Figure 5. MIC bioassay results of Streptomyces isolates against Penicillium digitatum.Clockwise from top: Streptomyces strain 328 after 11 days, the Streptomyces after 19 days, the Streptomyces after 20 days, blank agar hole (control).

Table 1 .
Antifungal activity of Streptomyces isolate 328 metabolites and culture against P. digitatum and P. italicum.

Table 2 .
Inhibition (%) produced by of Streptomyces isolate 328 metabolites from YMG and starch casein media against P. digitatum and P. italicum.

Table 3 .
Bioassay results of solubility tests of the antifungal principle(s) of Streptomyces isolate 328 against Penicillium digitatum in fractions of different solvents indicated by well diffusion-method at 10 mg ml -1 of dry crude.
Trejo-Estrada et al. (1998)7)olites assayed in solid medium tests presented a slight inhibition against Fusarium solani, but it was moderate to strong against Phytophtora megasperma.Streptomyces isolate No. 328 metabolites, in in vitro assays.Similar results of disease inhibition produced by Streptomyces sp.R03 metabolite against lemon pathogens has been reported earlier byMaldonado et al. (2010).Plaza et al. (2004)pointed out that it is not possible to extrapolate all in vitro results to natural systems since in vivo there are other factors, those in the environment as the nature of its factors, interfere with other microorganisms, etc. Streptomyces 328 metabolites was not inhibited by heat and choloroform; this effect could be due to their chemical nature and could not inhibit the lytic enzymes activity in fermentation broth.Mahadevan and Crawford (1997)andTrejo-Estrada et al. (1998)founded that, although antibiosis is one of the main mechanisms found in Streptomyces, the production of lytic enzymes (chitinases, β 1-3 glucanases) capable of acting on the fungus cell wall by altering growth and possibility is also common among them.Streptomyces isolate No. 328 metabolites have molecular mass higher than 2000 and their mode of action is fungistatic, but several authors report Streptomyces ability to produce fungicidal substances like Streptomycin, Kasugamycin, Polyoxin and Validamycin that have different action modes and the active ingredients of many biological plaguicides (Duran