Biocontrol of Alternaria alternata YZU, a causal of stem end rot disease on pitaya, with soil phosphate solubilizing bacteria

. Stem end rot is the most destructive disease caused by Alternaria alternata YZU in pitaya-growing regions of Vietnam. This study was conducted to characterize antagonistic phosphate-solubilizing bacteria (PSB) from rhizosphere soil for their biocontrol activities against A. alternata YZU and evaluate the effect of temperature, pH, and water activity on that antagonism. Among seven PSB isolated from 45 rhizosphere soil samples, PSB31 (identified as Bacillus sp . strain IMAU61039, Accession number: MF803700.1) exhibited the highest antagonistic activity against A. alternata YZU with an average inhibition diameter of 0.65 ± 0.05 cm. The results also show that the strain PSB31 controlled the mycelial growth of A. alternata YZU by secreting antifungal metabolites. The most potent inhibitory activity was identified under in vitro conditions of 25 °C, pH 7, and a w 1. The isolated PSB31 could be a potential biological control agent against A. alternata YZU.


Introduction
Pitaya (Hylocereus Undatus) is an important fruit crop grown well in Binh Thuan, Vietnam. In this region, however, there is a risk of fungal pathogens, particularly stem end rot caused by Alternaria alternata YZU, resulting in severe economic losses [1]. The control of fungal diseases attacking pitaya has been primarily performed by using chemical pesticides that are hazardous to human health, pollute the environment, and induce the resistance of phytopathogenic agents [2]. Hence, developing alternative methods to control this disease is crucial. Biological control with soil bacteria has received much attention as one of the non-hazardous pest management techniques against different plant pathogens [3].

Evaluating the antagonistic activity of isolated PSB
The potential for biological control of seven where R and r are the radial distance of the fungal pathogen growth for the control and the dual culture (mm) [1].  [13].

Effect of water activity
The water activity (aw) is defined as the ratio of the to PDA [13]. Glycerol attaches to a part of water, making it unusable for microorganisms. The dual culture was carried out. The PIRG was calculated after seven days of incubation in the dark at 25 °C.
The PDA medium was buffered to the desired pH with different buffers and adjusted by adding HCl or NaOH. The inoculated dishes were incubated at 25 °C. The PIRG was calculated after seven days of incubation.

Statistical analysis
All experiments were conducted in triplicates. The data were subjected to analysis of variance with the software STATISTICA for Windows v.6. The statistical significance of the results was determined by performing a test of Duncan's multiple ranges (p < 0.05). The results were expressed as mean ± standard deviation.

Antagonistic activity of bacterial strains
In  and selected for further studies.

Antifungal activity of PSB31 culture filtrates
The strain PSB31 significantly inhibited the mycelial growth of A. alternata YZU (

Effect of temperature on antagonistic activity of PSB31
All studied temperatures exhibited significant inhibition of PSB31, with the highest one (54.44%) at 25 °C (Fig. 2).

Effect of water activity on antagonistic activity of PSB31
The water exhibited significant inhibition against the mycelial growth of A. alternata YZU. The highest inhibition (71.23%) was found at aw equal to 1. At lower water activity values, the inhibition was also lower, indicating that PSB31 plays an essential role in inhibiting the mycelial growth of A. alternata YZU, and radial growth inhibition of the phytopathogenic agent increases with water activities (Fig. 3).

Effect of pH on antagonistic activity of PSB31
The in vitro assay of pH revealed that the pH significantly affected the antagonistic potential against A. alternata YZU (Fig. 4). pH 7 had the highest inhibitory effect on the growth of A. alternata YZU (51.14%); and the lowest effect (around 21%) was observed at pH 4, 4.5, and 9.

Discussion
Biological products for controlling plant diseases have been replacing fungicides. Stem end rot caused by A.
alternata YZU is one of the most destructive diseases that threaten pitaya production in Vietnam and worldwide. In this study, isolated rhizosphere PSB were evaluated for biocontrol activities against A. alternata.
Numerous studies have been carried out to identify different PSB strains and their efficacy in phosphate solubilization and to exploit the advantages of PSB for biocontrol activities and biofertilizers [16,17]. In addition, our data also showed the culture filtrate from the PSB31 strain significantly interfered with the growth of A. alternata up to 62.92%. A similar property was observed in B. siamensis strains LZ88 [15] and B. megaterium [19]. The culture filtrate of these strains was reported to inhibit mycelial growth, spore germination, and spore production in A. alternata [15,19]. This indicates the secretion of antifungal metabolites by the bacterial antagonist, such as hydrolytic enzymes, bacteriocins, antibiotics, or some other secondary metabolites [15,20].
On the other hand, it is well known that bacteria may also be good  [7]. Hence, our study provides evidence that the PSB31 strain influences the survival of A. alternata.
Biological control is essential in controlling the phytopathogen in sustainable agriculture [21]. Bacillus sp., such as B. subtilis and B. amyloliquefaciens, have been used in commercial products for biocontrol of plant disease because of their potential for biocontrol and high stability under harsh environmental conditions caused by spore forms [22]. In this study, we found that antagonists and host plants [24,25].  [7,14]. In this study, the bacteria antagonist showed the inhibitory effect at all temperatures studied, and the most potent inhibition in the fungal radial growth was observed at 25 °C.
Hence, the results agreed with those of previous studies [7,14].
Regarding water availability, the results showed that water activity significantly affected mycelial growth, which was optimal between 0.95 and 1. It is also consistent with some previous studies on Bacillus sp. For example, the optimal growth of B.
amyloliquefaciens was at a water activity of 0.960 and 37 °C [13], but it inhibited the growth of A. flavus and F.
verticillioides at a water activity of 0.99, 0.97, 0.95, and 0.93 [26] and also inhibited the growth and aflatoxin B1 production by Aspergillus section Flavi at a water activity of 0.982 [27]. The results suggested that changes in water activity might be useful for improving the environmental competence of the microorganism in the environment.
As for the influence of pH on the biocontrol, the results revealed that the pH of the growth medium played a crucial role in the inhibition competence of organisms, which was low at acid pHs, increased by increasing pH values to 7 (neutral pHs), and then decreased in alkaline pHs. Previous studies demonstrated that pH 6 to 7 was optimum for the growth and inhibition ability of the majority of bacterial strains [28,29]. We found that pH 6.5 was required for the optimum growth of PSB31. Therefore, the inhibitory effect of PSB31 against A. alternata YZU was investigated at pH 4.0-9.0. At pH 7.0, more than a 50% inhibition of the radial growth of A. alternata YZU was obtained, which decreased nearly to thirty per cent at pH 8.0 ( Figure 4). Hence, the result indicated that the optimal pH for antagonistic activity was 7. A higher or lower pH considerably reduced the growth and the antagonistic activity of PSB31 against A. alternata YZU.

Conclusion
Seven PSB isolated from rhizosphere soil samples could inhibit the mycelial growth of Alternaria alternata. The