Strawberry (Fragaria x ananassa (Weston) Duchesne ex Rozier) is an important fruit crop, grown in Morocco in the areas of Souss, Gharb and Loukkos. A major constraint to the culture of strawberry is the low tolerance of this species to fungal diseases  . The number of phytopathogenic fungi attacking this culture is vast, more than 50 genera  , resulting in severe economic losses. Fungal diseases can affect all parts of strawberry, but there are those who produce the crown rot, resulting in death of the plant, as anthracnose caused by Colletotrichum spp.    , especially C. acutatum, considered among the more devastating phytopathogens. The control of fungal diseases attacking strawberry plants is mainly done by treatment of the soil and the plants using chemical pesticides. The intensive use of fungicides leads to the accumulation of toxic compounds potentially dangerous for humans and the environment, as well as in the induction of the resistance of phytopathogenic agents    . Biological control has received great attention as one of the non-hazardous pest management techniques against diseases caused by phytopathogenic fungi, including anthracnose  . The selection of the antagonists planned for the biological control of plant diseases usually involves examining a large number of microbial isolates to increase the probability of discovering a strain highly effective. The natural antagonists on the surfaces of the host are promising components of protection of organic crops  . Antagonistic bacteria such as Bacillus subtilis   , B. amyloliquefaciens  and Pseudomonas fluorescens  or fungi such as Trichoderma harzianum   or yeast such as Saccharomyces cerevisiae   have been found effective for the control of the anthracnose disease under controlled research conditions.
Some environmental factors can strongly influence the biological effectiveness of the antagonist against plant pathogens. Climate change and water conditions are among the crucial factors influencing microbial activity in natural systems  . Therefore it is reasonable to study the influence of temperature, pH and water activity on the in vitro antagonism of Bacillus amyloliquefaciens and Trichoderma harzianum. However, there is a lack of comparative information on the effects of these factors on potential biocontrol of B. amyloliquefaciens and T. harzianum against plant diseases.
The objective of this study is to evaluate the effect of temperature, pH and water activity on the antagonism of Bacillus amyloliquefaciens and Trichoderma harzianum to Colletotrichum acutatum.
2. Materials and Methods
2.1. Fungal Pathogen Strain
Strain Ca6 of Colletotrichum acutatum was isolated from naturally infected strawberry fruits presenting anthracnose symptoms. It was selected for its aggressiveness among several isolates found in different strawberry cultivars. C. acutatum Ca6 originated from fields of strawberry plants of Loukkos (Larache, Morocco), developed well in Potato Dextrose Agar and was incubated ten days in 25˚C ± 2˚C before use. The identification was carried out by macroscopic and microscopic observations of the isolates using determination keys   .
2.2. Isolation of Antagonistic Bacterial Strains
Nine bacterial strains were isolated by the method of serial dilutions from rhizosphere soil and roots of strawberry plants taken from various agricultural zones of the Loukkos region (Larache, Morocco), and identified by Hamdache et al.
Table 1. Identification of antagonistic strains of Bacillus amyloliquefaciens  .
 . A molecular identification revealed that the nine antagonistic bacterial isolates belong to the species Bacillus amyloliquefaciens. Strains at the beginning were noted by an arbitrary notation I1, I2, I3, I18, B3, B12, RA9 and RA12 (Table 1).
2.3. Fungal Antagonist Strain
Trichoderma harzianum (TR) strain was isolated from soil into PDA (Potato Dextrose Agar) plates using spread plate technique. Litter materials were cultured in PDA plates for the isolation. The TR strain was isolated into pure culture on PDA. The identification was carried out by macroscopic and microscopic observations  .
2.4. Effect of Environmental Factors
The potential of biological control of the nine strains of Bacillus amyloliquefaciens and the strain of Trichoderma harzianum was assessed. The inhibition of Colletotrichum acutatum Ca6 according to the variation of some factors (temperature, pH, activity of the water) was evaluated by calculating the percentage of inhibition of mycelial growth on Petri dishes by dual culture technique on PDA. The antagonist and the phytopathogen were put on the opposite sides of Petri dish at the same distance from the periphery. A completely randomized experimental device was used with three replicates for each antagonist.
2.4.1. Effect of Temperature
The inoculated dishes were incubated in the dark at 18˚C; 23˚C; 25˚C; 27˚C and 30˚C for 7 days.
2.4.2. Effect of Water Activity
The water activity (aw) represents the availability in open water for the biochemical reactions for the development of microorganisms. Different values of activity of the water have been tested (1; 0.95; 0.90 and 0.85) by the addition of glycerol in PDA   , which will attach a part of the water and make it unusable to microorganisms. The same technique of dual culture has been fol-
Table 2. pH values and buffers used  .
lowed. After seven days of incubation in the dark at 25˚C, the PIGR (Percentage of Radial Growth Inhibition) has been calculated.
2.4.3. Effect of pH
To assess the effect of pH on the inhibition of mycelial growth by antagonistic bacteria, the following pH, acid, neutral, and basic, have been tested: 4; 4.5; 5; 5.5; 6; 6.5; 7; 7.5; 8 and 9 (Table 2). The medium PDA has been stamped, according to the desired pH, by different buffers. Using a pH meter, the pH has been adjusted by addition of HCl to the pH acids and NaOH to the basic pH. The boxes were incubated at 25˚C. The PICR is calculated after seven days of incubation.
2.4.4. Percentage of Inhibition of Radial Growth
After the incubation period, the radial growth of the pathogens was measured and the percent inhibition (1) of mean radial growth was calculated relative to the control as follows:
PIRG: Percent Inhibition of Radial Growth of pathogen’s mycelium;
T: Radial growth of control agent;
C: Radial growth of the pathogen in the presence of the antagonist.
2.4.5. Statistical Analysis
All analyses were conducted in triplicates. The percent of inhibition of mycelial growth of the phytopathogenic agent by the antagonists have been subjected to an analysis of variance (ANOVA) using the software STATISTICA for Windows v.6. The statistical significance of the results was determined by performing a test of Duncan’s multiple range (p < 0.05). Results were expressed as mean ± standard deviation.
3.1. Dual Culture Technique
Bacillus amyloliquefaciens strains and Trichoderma harzianum provide inhibitory effects on the mycelial growth of the phytopathogenic strain (Figure 1). The inhibition of development of Colletotrichum acutatum isolate varies within B. amyloliquefaciens strains.
3.2. Effect of Temperature
The percent inhibition of mycelial growth by Bacillus amyloliquefaciens differs according the isolates and the temperatures studied. Isolate Bc2 is the most effective and presents the highest percent inhibition (Figure 2), whereas Bc5 has the lowest percent inhibition at all tested temperatures. Trichoderma harzianum
Figure 1. In vitro mycelial growth of Colletotrichum acutatum in PDA (a); dual culture between the antagonists and the phytopathogen in PDA (b, c and d); Inhibition of growth of C. acutatum by Trichoderma harzianum TR (b) and Bacillus amyloliquefaciens (isolate I2 (Bc2) (c) and isolate B3 (Bc4) (d)).
Figure 2. Effect of temperature on growth inhibition of Colletotrichum acutatum Ca6 by nine Bacillus amyloliquefaciens strains.
presents a very high efficiency on the inhibition of radial growth of Colletotrichum acutatum with an inhibitory effect exceeding 70% at all tested temperatures (Figure 3).
3.3. Effect of Water Activity
The inhibition of mycelial growth of Colletotrichum acutatum is important in the presence of the isolate Bc2 and low in the presence of Bc5 (Figure 4). Trichoderma harzianum has a high potential of radial growth inhibition of the phytopathogenic agent that increases with the water activity (Figure 5).
3.4. Effect pH
The pH presents a large effect on the antagonistic potential of Trichoderma harzianum (Figure 6) and Bacillus amyloliquefaciens (Figure 7). Among the bacterial isolates, Bc2 shows a large inhibitory effect on the growth of Colletotrichum acutatum; contrariwise, Bc5 has the lowest inhibitory effect (Figure 7).
Figure 3. Effect of temperature on growth inhibition of Colletotrichum acutatum Ca6 by Trichoderma harzianum.
Figure 4. Effect of water activity on growth inhibition of Colletotrichum acutatum Ca6 by nine Bacillus amyloliquefaciens strains.
Figure 5. Effect of water activity on growth inhibition of Colletotrichum acutatum Ca6 by Trichoderma harzianum.
Figure 6. Effect of pH on growth inhibition of Colletotrichum acutatum Ca6 by Trichoderma harzianum.
Figure 7. Effect of pH on the inhibition of radial growth of Colletotricum acutatum Ca6 by nine antagonistic bacterial strains.
Biological control is an alternative to the use of phytochemicals in involving biological products in the control of plant diseases. In this study, we found that Bacillus amyloliquefaciens and Trichoderma harzianum provide inhibitory effects on the development of Colletotrichum acutatum, the phytopathogenic agent of the anthracnose of strawberry (Fragaria x ananassa). Biological control of Colletotrichum species has been demonstrated in other studies using Trichoderma species  and Bacillus species  . This study shows that T. harzianum grows faster than C. acutatum Ca6 strain. This rapid growth suggests a mycoparasitism, at least in the experimental conditions tested, and gives Trichoderma an important advantage in the competition for nutrients and space with phytopathogenic fungi  . Biological control is important in crop production disease control   . Bacillus subtilis and B. amyloliquefaciens have been used in commercial biological control products due to their potential of biocontrol and high stability in harsh environmental conditions caused by spore forms  .
The environmental factors play an important role, since they affect the biological life of the microbial species and the physiology/metabolism of pathogen antagonist and host plant   . Several species antagonists of Bacillus spp. have shown efficiency in the fight against the anthracnose of multiple hosts      .
The inhibition of mycelial growth of Colletotrichum acutatum by the Bacillus amyloliquefaciens strains tested and by changing the temperature, water activity and pH, shows that the potential of biological control varies from an isolate to the other. Bacillus amyloliquefaciens isolates have a different effect on mycelial growth of the phytopathogen. Bc2 shows a great effect compared to other isolates, Bc5 has a weak influence on radial growth. Trichoderma harzianum has a large antagonistic effect on the mycelial growth of the phytopathogenic agent. Hamdache et al.  have worked on the same bacterial strains to fight against Botrytis cinerea and have found that strain Bc7 has an efficiency of upper control against Botrytis cinerea compared to other strains to the different conditions tested, while strain Bc4 is the least effective. For the control against Colletotrichum acutatum we found that the isolate Bc2 is the more efficient compared to other isolates and Bc5 the less effective to the different conditions tested.
B. amyloliquefaciens show good antifungal activity on various plant pathogens, can be effectively used for controlling phytopathogens including Colletotrichum acutatum  . Trichoderma harzianum also has a great potential of biocontrol against anthracnose caused by C. acutatum    .
Temperature has a great influence on the development of microorganisms as well as their biological activity. All bacterial isolates represent a large inhibitory effect of radial growth at 25˚C. Studies have been made on the influence of environmental parameters on the development of Colletotrichum acutatum and have found that the optimal values of the temperature is 25˚C ± 2˚C    , Bc2 is the most effective among the other isolates and Bc5 the least effective. The fungal antagonist has a great inhibitory effect at all temperatures studied, and rises by increasing the temperature. Trichoderma harzianum has high efficacy at 33˚C or lower against Sclerotium rolfii, another phytopathogenic agent, and produces secondary metabolites and mycotoxins at high temperatures that will help control plant pathologies  . Antagonistic activity in vitro of T. harzianum to B. cinerea is more effectively at a near-optimal 25˚C is consistent with a more rapidly increasing conidial respiratory rate at the higher temperature   . The optimum temperature for radial growth of Trichoderma spp. is between 25˚C and 30˚C  .
The water activity has a remarkable way on the mycelial growth, which is optimal between 0.95 and 1. The activity of water has also been favorable to inhibit the growth of Verticillium dahliae and Rhizoctonia solani by Trichoderma harzianum  . At 37˚C the optimal growth of B. amyloliquefaciens was at aw 0.960   , this antagonist has inhibited the growth of A. flavus and F. verticillioides at aw = 0.99; 0.97; 0.95 and 0.93  and inhibited the growth and aflatoxin B1 production by Aspergillus section Flavi at aw = 0.982  . Maximal growth rates of T. harzianum were observed at aw 0.997   , the optimal aw values for mycelial growth and in vitro enzyme activities were similar  . The in vitro enzyme activities of T. harzianum were also affected by aw, but significant enzyme activities were measured for most of the enzymes even at aw values less than the limit of mycelial growth  .
Bacillus amyloliquefaciens and Trichoderma harzianum were influenced by pH, which was low at acid pH, and increased by increasing pH values to 7 (neutral pH) and then began to decrease in alkaline pH. Colletotrichum musae, agent to anthracnose of the banana tree, develops at an optimal pH equal to 4.5  . The tolerance of B. amyloliquefaciens to grow under different pH-temperature was studied by Gotor-Vila et al.  ; they have found that the optimum growth was observed at 37˚C and pH 5-7  . B. amyloliquefaciens has exhibited significantly low activities of starch-degrading enzymes and high resistance to low pH  . The crude lipopeptides of B. amyloliquefaciens were insensitive to pH variation. The activity was not affected at pH 2 to 11, and was reduced at pH 12   which means variation of pH affect the antifungal activity of this antagonist. T. harzianum were able to grow on a wide range of pH from 2 to 6, and the optimal growth was observed at pH 4, the mycelial growth ceased at pH 8 and 7, also pH had an effect on the in vitro of enzymes activities of T. harzianum  . Jackson et al.  have found that optimum biomass production Trichoderma harzianum occurred at pH 4.6 - 6.8.
The antagonists behave differently depending on the environmental parameters. We found that isolate Bc2 show more efficiency compared to other bacterial strains, and that Trichoderma harzianum always inhibits the development of the pathogen despite the change in the factors studied. However, other parameters could be considered to develop and improve the control efficiency by these antagonists in order to acquire a viable biological control system against the brown spot disease caused by Colletotrichum acutatum. Adaptation of biocontrol potential of the antagonists studied to environments with different temperature; aw and pH characteristics seems to be an important mechanism of evolution enabling the effective competition for nutrients under a wider range of these environmental parameters.
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