Control of Alternaria Leaf Spot of the Common Bean (Phaseolus vulgaris L.) Using Soil-Derived Biological Agents

Phaseolus vulgaris L. is considered one of the most essential legume crops in Kenya. Alternaria alternata is an economically significant plant pathogen that causes Alternaria leaf spot which accounts for over 70% yield losses of beans in Kenya. Chemical fungicides based on copper and sulfur are used to control Alternaria leaf spot in bean plants, but their prolonged use has adversely affected the environment and the health of workers. Herein, we tested the biocontrol potential of bacterial agents from soil planted with Rosecoco bean plants infected with A. alternata. Using bacterial suspensions at different time intervals, we evaluated the putative bacterial biocontrol activity against A. alternata under greenhouse conditions. B. subtilis and B. velezensis bacterial biocontrol agents significantly suppressed disease severity by 20% and 21.2% on the 45th day, respectively. Our study demonstrates that B. subtilis and B. velezensis are promising biocontrol agents that could be integrated in the management of Alternaria leaf spot.


Introduction
Te common bean Phaseolus vulgaris is a crop that is planted worldwide.In Kenya, approximately 75% of this crop is grown in the Eastern, Nyanza, and Rift Valley regions which are characterized by well-drained soils, low-altitude areas, with 800-2000 mm rainfall, and an optimum temperature of 20-25 °C [1].GLP X92 (Mwitemania), GLP X1127 (Mwezi Moja), and GLP 2 (Rosecoco) are the varieties that are predominantly grown in these regions [2].Low soil fertility arising from poor farming systems and biotic factors such as bacterial and fungal diseases are a major challenge to smallholder farmers [3].Among the fungal diseases that lower the common bean yield in Kenya is Alternaria leaf spot disease caused by the fungus Alternaria alternata [4].
Alternaria alternata attacks bean leaves causing characteristic small irregular brown lesions which can turn black with centered rings [5].Spotting occurs on lower and mature leaves, and the lesions may dry and fall of causing a shothole appearance on the leaf when the infection is severe or combined to form huge necrotic spots [6].Heavily spotted leaves may become yellow, wither, and fall due to water defciency [5].Management of Alternaria leaf spot in Kenya is carried out using sulfur-and copper-based fungicides like Bonide ™ , Cuptocafaro WP, and Cuprocafaro Micro 37.5 WG, respectively [7].Sulfur fungicides prevent the spread of fungal spores, and copper ions kill the fungus by denaturing the enzymes and proteins in pathogen cells.Although the sulfur-and copper-based fungicides have been efective in minimizing the spread of Alternaria leaf spot, they irritate the applicants' eyes and skin, can cause bodily harm if ingested, and also pollute the environment [8].
Biological control has been postulated as an alternative method for controlling Alternaria leaf spot because it is ecofriendly and less toxic as compared to synthetic chemicals.Management of disease-causing macroorganisms using rhizobacterial strains has previously been reported [9,10].Diverse species groups of microorganisms colonize plant tissues and provide long-term protection throughout the growing period [11,12].Disease suppression occurs when the colonizing microorganisms compete for space or nutrients with the pathogen or produce lytic enzymes, siderophores, and antibiotics [13,14].Tozlu et al. [15] reported the potential application of Trichoderma harzianum and Bacillus species isolated from the rhizosphere of tomato plants in controlling A. alternata.We hypothesized that some of the microbes within the bean rhizosphere microbiome could have the potential to suppress soil fungal pathogens.In Kenya, these soils are yet to be explored for their potential biocontrol application.Terefore, the aim of this study was to isolate and screen for bacterial antagonists for A. alternata that infects common beans.

Isolation and Characterization of Fungal Pathogen and
Bacterial Biocontrol Isolates.Purposive sampling was conducted at one of the universities of Embu farm plots (0.30′47.0″S 37 °27′09.46″E)using GLP 2 bean plants displaying leaf spot symptoms.Leaves were collected and surface-sterilized using the following protocol: 3% bleach plus Tween 20 for 30 seconds, 70% ethanol for 10 seconds, and a thorough rinse using sterile water for 30 seconds.Te leaves were cut into thin sections using sterile scalpel blades, macerated in sterile phosphate-bufered saline (PBS), and vortexed to homogenize the solution.Te solution was serially diluted, and 100 μL aliquots from dilutions of 10 − 1 and 10 − 2 were plated onto potato dextrose agar (PDA plates) amended with ampicillin (50 μg/mL) to inhibit the growth of bacteria.Te plates were incubated at 30 °C for 7 days to allow fungal growth.Mycelia were picked based on morphological features and transferred to new PDA plates to obtain pure cultures.We sampled soils from the same bean growing plots because we hypothesized that these soils contain microbes that could suppress fungal pathogens.One gram of soil was mixed with 1 mL of PBS and serially diluted, and 100 μL from dilutions 10 − 2 , 10 − 4 , and 10 − 6 was spread plated onto Trypticase soy agar plates supplemented with cycloheximide (50 mg/L) to inhibit the growth of fungi and incubated at 30 °C for 24 hours.Morphological characteristics such as color, elevation, and form were examined using standard microbiological techniques and recorded as described [16].Distinct colonies were selected and transferred to new plates to obtain pure cultures.

Antagonism Assays of Bacterial Biocontrols.
Single colonies were selected, purifed, and screened for ability to inhibit A. alternata as follows: Ten microliters of fungal culture broth containing 1 × 10 5 spores was spread plated onto nutrient agar plates, and 10 μL of the bacterial biocontrol was spotted using a sterile pipette.Te plates were incubated at 30 °C for 24 hours.Zones of inhibition around the bacterial colonies indicated antagonistic activity against A. alternata.Te inhibition zones measuring 1.5-3.5 mm were considered signifcant and indicated possible biocontrol agents for A. alternata.

Biochemical and Physiological Characterization of Bacterial
Isolates.Te bacterial isolates were then tested for their ability to utilize starch, carboxymethyl cellulose, cellulose, skimmed milk, tributyrin, citrate, and phosphate polymers by spotting them on nutrient agar media supplemented with respective substrates.Te plates were incubated at 30 °C for 24 h.Carboxymethyl cellulose and starch plates were fooded with Gram's iodine solution.Formation of clear zones around bacterial growth after staining showed positive enzyme production, while the presence of blue-black color on starch plates indicated the absence of enzyme production.Lipolytic activity was indicated by clear zones around the tributyrate agar, while the absence of clear zones indicated negative results.Citrate utilization was tested through introducing isolates into Simmons citrate agar slants and incubating them at 30 °C.Media change from green to blue showed positive results, while media retaining green color indicated negative results.For phosphate utilization, bacterial isolates were inoculated onto media containing phosphate supplemented with bromothymol blue and incubated at 30 °C for 48 hours.Formation of clear zones around the bacterial isolates and media color change from green to orange indicated positive results.To understand the optimum growth conditions, the bacterial biocontrol agents were tested for optimum growth at salt concentrations of 5-15%, pH ranges of 5-12, and temperature ranges of 25− 45 °C.Growth was assessed by measuring OD 600.

Molecular Characterization of Fungal Pathogen and
Bacterial Biocontrol Isolates.Genomic DNA was extracted using the phenol-chloroform protocol as described by [17] and used as a template for polymerase chain reaction.PCR amplifcation of the ITS region was performed using the universal fungal primer pair of ITS1 (5′-TCCGTAGGTGAA CCTGCGG-3′) and ITS4 (TCCTCCGCTTATTGATATGC-3′) [18].Amplicons were generated on Sure Cycler 8800 (Agilent Technologies) in a total reaction volume of 30 μL (22 μL PCR water, 6 μL of polymerase bufer, 0.6 μL of 20 μM ITS 1 primer and 0.6 μL of 20 μM ITS 4 primer, 0.6 μL Taq polymerase (0.5 μM), and 0.2 μL of 50 ng genomic DNA).Te PCR conditions were as follows: 45 sec of denaturing at 95 °C, 30 sec of annealing at 53 °C, and 45 sec of elongation at 72 °C followed by a last step of 4 min at 72 °C.Amplifed products were separated on 1% agarose gel in EDTA bufer and visualized under UV light after staining with a fuorescent dye.Te amplifed fragments were cleaned by mixing 12.5 μL of the PCR product with 2.5 μL of Exo SAP-IT (Termo Fisher Scientifc) and incubated at 37 °C for 30 min followed by heating the mixtures at 95 °C for 5 min to stop the reaction [19].Genomic DNA from the bacterial isolates was extracted and purifed by the method described by Chaudhary et al. [20].Te 16S rRNA gene was amplifed by primers (27F and 907R) using Sure Cycler 8800 (Agilent Technologies).30 μL master mix comprising 22 μL PCR water, 6 μL of 5 ×Taq bufer, 0.6 μL of Taq polymerase (0.5 μM), 0.6 μL of 10 μM reverse primer, 0.6 μL of 10 μM forward primer, and 0.2 μL of 50 ng DNA were subjected to 35 cycles of initial denaturation at 95 °C for 5 minutes, strand thickening at 45 °C for 30 seconds, elongation at 72 °C for 45 seconds, and a containment step at 10 °C [20].Te PCR amplicons were separated using 1% agarose gel and visualized under UV after staining with a fuorescent dye.Te amplicons were sequenced at Inqaba Biotech, South Africa.

Scientifca
Sequences obtained were edited using Chromas Lite software.Basic Local Alignment Search Tool (BLAST) in the National Center for Biotechnology Information (NCBI) website was used to compare the 16S rRNA gene sequences with those in the NCBI database in order to know the closest relatives as described by Al-Jaradi et al. [21].Te evolutionary history was inferred using the neighbor-joining method, and the evolutionary distances were computed using the maximum composite likelihood method.Evolutionary analyses were conducted in MEGA11.

Evaluation of Putative Bacterial Biocontrol Activity against A. alternata under Greenhouse Conditions.
Te experiments were conducted in a randomized complete block design of three blocks with ten bacterial biocontrol treatments and respective positive and negative controls.Tree GLP 2 Rosecoco bean seeds were planted in twelve pots, and each treatment was replicated three times.Te bean seeds were watered every seven days.Two hundred milliliters of bacterial biocontrol suspensions was prepared by diluting a 24 h bacterial culture to a cell concentration of 1 × 10 8 CFU/mL and sprayed to runof on the upper and lower leaves of 21day-old bean plants.Tree hours later, an A. alternata spore suspension containing 1 × 10 6 spores/mL was spotted on the second, third, and fourth leaves using sterile cotton buds.Te positive control plants were sprayed with a commercial Bonide sulfur fungicide according to the manufacturer's instructions, while the negative control plants were sprayed with sterile distilled water following A. alternata application.We sprayed the plants with biocontrol suspensions on the 15th, 30th, and 45th day postinfection with A. alternata.Disease incidence was recorded as the percentage of diseased leafets collected per treatment.Disease severity was scored and recorded using the leaf symptom index (LSI) at 15, 30, and 45 days beginning at ffteen days after inoculation using a 0-5 rating scale as follows: 0 � no symptoms in all leaves, 1 � only 1 leaf partially wilted, 2 � 2-3 wilted leaves, 3 � 2/3 wilted, 4 � all leaves wilted, and 5 � plant dead.Te lesions were classifed as follows: no lesions � 1, lesions >30% � 2, lesions >60% � 3, lesions >90% � 4, and plant death � 5 [22].A total of 3 LSI estimations were made per treatment.Leaf disease incidence and leaf numbers were determined at the close of the feld experiment.Analysis of variance was used to assess the efectiveness of bacterial biocontrol against A. alternata, and the mean diferences between controls and diseased plants were compared using Duncan's multiple range test at P � 0.05.To evaluate the colonization efciency of the bacterial biocontrol and pathogen, we reisolated and characterized both using standard microbiological procedures as described above.Te experiment was repeated twice under the greenhouse conditions of 27-30 °C.

Morphological Characterization of A. alternata.
Five unique fungal isolates were obtained from diseased leaf samples.All the fungal isolates grew within one week at 28 °C and showed diferent morphologies (Figure 1).

Morphological Characterization of Bacterial Biocontrol
Isolates.Of twenty-seven bacterial isolates recovered, eight showed antagonistic activity against A. alternata, and these were selected for further analysis.Morphological characterization was performed on the basis of color, elevation, form, and margin (Table 1).

Physiological and Biochemical Characterization of Bacterial Isolates.
Tese isolates could grow between pH 4 and 12 with the optimal pH recorded at pH 6 except isolate U5 (pH5).Growth decreased when pH was above 8 (Figure 2(a)).All isolates were able to grow within temperatures of 25-45 °C.Te optimum temperature for the isolates was 25 °C with the least growth at 45 °C (Figure 2(b)).Te optimum salt concentration for the isolates was 5%, while growth decreased towards 30% (w/v) as shown (Figure 2(c)).
Te isolates grew on various substrates as shown (Table 2).All isolates utilized starch as a sole carbon source, while isolates C16 and C1c utilized all substrates.

Molecular Characterization of Fungal and Bacterial
Isolates.A total of 26 fungal isolates were isolated from diseased leaves of the GLP 2 Rosecoco bean variety.BLAST analysis showed that 5 of them were afliated to the genus Alternaria with varying levels of relatedness (Table 3).
Eight bacterial isolates were identifed as having antagonistic activity against the fungal isolates (Table 4).
Te isolates were closely clustered to the genus Myroides, Bacillus, Paenibacillus, and Brevibacillus, and they showed activity against A. alternata with some showing strong clear zones and other moderate or weak clear zones (Figure 3).
Te highest disease incidence was recorded at 15 days for Bonide sulfur-treated plants and untreated plants (Figure 5).Te lowest disease incidence was recorded at 45 days for plants treated with B. subtilis and B. velezensis (Figure 5).At 30 days, the highest disease severity was recorded for Bonide sulfur-treated plants and untreated plants, while the least disease severity was recorded for plants treated with Scientifca B. subtilis and B. velezensis.Tere was a signifcant diference (P > 0.001) in disease incidence and severity among the bacterial biocontrol treatments and the untreated plants (Figures 4 and 5).

Te Efect of B. subtilis, Positive Control (Bonide Sulfur), and Negative Control (Untreated Plants) on GLP 2
Bean Leaf Appearance under Greenhouse Experiment.B. subtilis-treated leaves were not spotted, Bonide sulfurtreated leaves were moderately spotted with light brown spots and yellowing, while untreated leaves were heavily spotted turning with light brown centers surrounded by dark concentric rings and yellow halos as shown in Figure 6.

Reisolation and Molecular Characterization of Fungal
Pathogen and Bacterial Biocontrol Isolates.Bacterial and fungal colonies were reisolated from GLP 2 bean leaves which were surface-sterilized and cut into tiny pieces of about 2-3 mm.Leaf material was crushed and suspended in PBS, and serial dilution was conducted.One hundred microlitres of dilutions 10 − 2 and 10 − 4 was plated on PDA and TSA plates for fungal and bacterial growth, respectively.Tree bacterial isolates and two fungal isolates were reisolated and identifed as shown (Table 5).

Discussion
Tis study explored the potential of isolates recovered from the rhizosphere to control brown leaf spot disease in GLP 2 bean leaves.By using cultural and molecular techniques, we determined that A. alternata was the causative agent of the brown spots on diseased GLP 2 bean leaves.We hypothesized that rhizospheric bacteria present in bean felds could have biocontrol properties.We characterized seven unique biocontrol isolates identifed as B. subtilis, B. velezensis, B. amyloliquefaciens, M. odoratimimus, B. laterosporus, P. polymyxa, and P. peoriae based on their ability to inhibit the growth of A. alternata.B. subtilis and B. velezensis showed strong inhibitory efects.Tese fndings are in agreement with those of Govardhani et al. [23] who reported that B. subtilis reduced A. alternata radial growth, while Toral et al. [24] reported that B. velezensis suppressed in vitro growth of pathogenic A. alternata by about 40%.According to Desmyttere et al. [25], Bacillus spp.produce several lipopeptide metabolites that adhere to and disrupt target fungi's cell membranes.In vitro, Bacillus spp.produce hydrolytic enzymes like lipases and proteases that break down fungi's cell walls [24].
Te isolates had the highest growth at pH 5.0-6.0 and decreased growth at pH values 7, 8, 9, and 10.Enzymatic activity increases up to the optimum pH and decreases beyond optimum pH.Drastic variation in pH can destroy the plasma membrane of bacteria and prevent enzyme activity and membrane transport proteins, thus decreasing growth.Tis fnding is in line with Trimulyono [26] who reported that most Bacillus spp.isolated from the soil are neutrophils growing optimally at one or two pH below pH 7. At optimum temperatures, all biocontrol isolates grew best at 25 and 30 °C and least at 45 °C.Metabolic activity increases at optimum temperatures [26].Te bacterial enzymes become denatured at temperatures above 45 °C, resulting in a decrease in growth.Based on this, it is likely that our biocontrol isolates are mesophiles, which grow best at  4 Scientifca a temperature of 25 °C.We observed that the ability of our biocontrol's to withstand a range of 10 and 15% NaCl indicated that they are slightly halophilic.Eight out of ten biocontrol agents with antagonistic efects on agar plates were similarly efcient in reducing leaf disease severity and incidence in GLP 2 bean plants.Our data showed that rhizospheric bacteria could serve as biocontrol agents for Alternaria leaf spot instead of commercial Bonide sulfur.According to Trimulyono [26], Bacillus spp.are a potential bioagent against Alternaria fruit rot.Te bacterial bioagents inhibit pathogen development by producing antibiotics or enzymes, competing with pathogens, and colonizing rapidly.In addition, Lastochkina et al. [27]

Conclusion
Of eight identifed bacterial biocontrol isolates, rhizospheric B. subtilis and B. velezensis displayed the highest antagonistic activity against A. alternata.Te two Bacillus species could be excellent candidates for ofering long-term biocontrol potential for the management of Alternaria leaf spot in GLP 2 bean plants.Future studies should combine biocontrol agents to test their efcacy against Alternaria leaf spot.

Figure 1 :
Figure 1: Morphological characteristics of select fungal isolates.(a) A. alternata isolate dark grey in color; (b) A. alternata isolate light grey in color; (c) Alternaria spp.isolate white in color; (d) Alternaria spp.isolate light brown in color.

Figure 3 :
Figure 3: Antagonistic activity of selected bacterial isolates against A. alternata.

Figure 4 :Figure 5 :
Figure 4: Disease severity of Alternaria leaf spot in GLP 2 Rosecoco bean variety plants at 15, 30, and 45 days postinfection.Values followed by the same values are not signifcantly diferent (P < 0.001).

Figure 6 :
Figure 6: Pot experiment showing the efect of B. subtilis, positive (Bonide sulfur), and negative control (untreated) on GLP 2 bean leaf appearance.(a) B. subtilis-treated leaves were not spotted; (b) Bonide sulfur-treated leaves were moderately spotted; light brown spots with a yellow halo around them; (c) untreated leaves were heavily spotted with light brown centers surrounded by dark concentric rings and yellow halos.

Table 1 :
Morphological characterization of bacterial biocontrol isolates.
found that harvested tubers treated with

Table 2 :
Substrate utilization by the various bacterial isolates.

Table 3 :
Phylogenetic analysis and identifcation of fungal isolates isolated from leaves of bean plants displaying leaf spot symptoms.

Table 4 :
Antagonistic activity of the bacterial isolates against A. alternata.