Influence of Inoculum Concentration on In Vivo Incubation Period of Emmia lacerata, Pathogenesis and Management of Wilt in Pepper (Capsicum annuum L.)

Experimental Sites and Sources of Microbial Strains

The in vitro and screenhouse experiments were carried out between February - April, 2023 and July – September, 2023, respectively. The experiments were conducted at a Research-Extension Unit of the Federal College of Agriculture, Ibadan (7°25’N; 3°50’E). Molecular identification of microbial strains was done at Bioscience Laboratory of the International Institute of Tropical Agriculture, Ibadan.

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Effect of Inoculum Dose on Pathogenicity and Incubation of Emmia lacerata in Pepper

This was conducted to investigate the influence of spore concentration on disease establishment and severity of wilt in pepper. Such information will be necessary for soil diagnosis before cultivation, as well as subsequent investigation on the impact of soil fungal population on accumulation of E. lacerata, especially, in asymptomatic hosts with medical significance.

Sandy-loam soil samples were collected from the Bora Farm of Federal College of Agriculture, Moor Plantation, Ibadan (7°22’32.0”N; 3°50’49.7”E) and subsequently steam- sterilised. Seeds of C. annuum (acquired from the Seed Store of the Institute of Agricultural Research and Training, Ibadan) were surface-sterilised with 2% NaOCl, and sown in 7 kg steam-sterilised potted soil. Emmia lacerata was cultivated in Potato Dextrose Broth (PDB) for 3 days at 30°C to generate spores. The rhizosphere of 10-day-old pepper seedlings was inoculated with 1 mL of different inoculum doses (106, 107 and 108 spores/mL) of the pathogen [14]. Infected seedlings were observed for 6 weeks after sowing to determine the period between inoculation and manifestation of wilt symptoms (in vivo incubation duration). Disease severity rating and percentage severity index were determined using the severity scale described by Bindal S, et al. [15] as 0 = healthy plant to 5 = dead plant stump.

R Ip Wilt severity index 100 T Sh ×   = ×   ×   ∑

Where R = severity score; Ip = number of infected plants; T = total number of plants under consideration, and Sh = highest severity score (5).

In vitro Inhibitory Assay of Bacillus Strains Against Emmia lacerata

Characterization of antifungal efficacy of biocontrol strains against mycopathogens is an essential step to select inhibitory Bacillus species for subsequent in vivo studies.

The antimycotic property of selected Bacillus strains against E. lacerata was determined through a dual-culture confrontation assay, as described by Cao Y, et al. [16]. The growing edge (5 mm) of 5-day-old E. lacerata was cut using a cork-borer and inoculated at the centre of PDA in a Petri dish. After 24 hours of incubation at 30°C, a day-old strain of each Bacillus species was spot inoculated on the plate, 3.5 cm away from the centre (containing the E. lacerata) [16]. Positive control plates (without Bacillus) and plates with dual cultures were incubated for 6 days at 30°C. Mycelial control was determined using the formula described by Hammami R, et al. [17]:

( ) MD Mc

Mycelial inhibition %

$$ ) = \frac {\mathrm {M D} - \mathrm {M c}}{\mathrm {M D}} \times 1 0 0 $$ Where MD = mycelial diameter on control plate and Mc = mycelial diameter on dual culture plate.

Effect of Inoculum Dose on the Biocontrol of Emmia lacerata with Bacillus Strains

One of the factors affecting the adoption of commercially available biocontrol materials in crop production is the lack of graduation, targeted towards specific pathogens. The efficacy of whole-cell biocontrol strains could be influenced by population of soil pathogens. Hence, depending on the outcome of initial soil diagnosis, a dose-dependent management procedure may be necessary. To determine the effect of inoculum dose on Emmia wilt management.

The rhizosphere of 10-day-old pepper seedlings was inoculated with 1 mL of different inoculum doses (106, 107 and 108 spores/mL) of the pathogen [14], as earlier described. Twenty-four hours after seedling inoculation with the pathogen, rhizosphere of hosts was treated with each Bacillus strain (108 cfu/mL). The experiment was arranged in a completely randomized design containing 3 concentrations of inoculum and two Bacillus strains. Positive control plants were uninoculated and negative (infected) control plants were treated with water. The treatments were replicated 4 times and the influence of inoculum size on wilt-biocontrol potential of selected Bacillus strains was determined 4 weeks after plant inoculation (6 weeks after sowing). Disease severity was established as illustrated by Bindal S, et al. [15], while percentage wilt control (PWC) was determined as described below.

DSC DST Wilt control 100 DSC − = ×

Where DSC = wilt severity index of negative control plant and DST = wilt severity index of Bacillus-treated plant [18].

Effect of Inoculum Concentration on Pathogenicity and Incubation Duration of Emmia lacerata in Pepper

Emmia lacerata FMIB29 caused wilt disease, through rhizosphere inoculation, in pepper plant. Manifestation of leaf chlorosis (Figure 3) was observed in infected seedlings 2 weeks post-inoculation with different concentrations of E. lacerata. The period between plant inoculation and the expression of wilt symptoms (2 weeks) was regarded as the incubation period for the pathogen in infected pepper seedlings. Chlorotic plants gradually became necrotic, and this resulted in crown wilt. Differences in inoculum dose, as used in this experiment, neither affected the incubation period observed for E. lacerata in pepper nor the severity of the disease (Table 1). Although, at 6 after sowing, plants inoculated with 108 spore/mL expressed the highest wilt disease severity index (45.01%), this was not significantly higher than the severity observed in plant sets inoculated with lower pathogen concentrations (107 and 106 spore/ mL). Control plants, treated with sterile distilled water, in place of Emmia species, expressed no wilt symptom through the course of the test.

As previously observed in tomato plants [8], the phytopathogenicity of E. lacerata was demonstrated (in pepper) in this study. However, the medical significance of E. lacerata, a fungus previously reported as wood-decaying organism, was established by Lee J, et al. [9]. The organism was described as an emerging human pathogen associated with fungal pneumonia. Supportive clinical pathogenicity assessment included chest consolidation, nodular opacity, air-fluid level, pleural effusions and cavitation. In contrast, the symbiotic potential of E. lacerata strains (as biocontrol agents) was investigated against multiple phytopathogenic fungi and oomycetes [19]. Emmia lacerata HG2011 was reported to successfully control eggplant stem-blight and cucumber vine-blight in infected plants. The occurrence of different physiological forms of E. lacerata might be as a result of horizontal gene transfer between pathogenic and non-pathogenic strains of the species [20].

Mobile pathogenicity chromosomes can be transferred between different morphological forms of the same or different microbial species. A typical example of interspecific virulence gene transfer was demonstrated between Stagonospora nodorum and Pyrenophora triti-ci- repentis [21]. However, occurrence of intraspecific, mobile pathogenicity-chromosome transfer was established between non-pathogenic and virulent strains of Fusarium species [22]. Such transfer of virulence conferred on the non-pathogenic strains the ability to infect tomato host. Consequently, the awareness of physiological forms with different host specificity is imperative in the application of E. lacerata as endosymbiont or as biocontrol agent against phytopathogens. This would guide against the possibility of cross-infection (especially, in immunocompromised human hosts) caused by the consumption of fruits produced by infected or inoculated plants.

Concentration of Emmia inoculum appeared not to significantly affect the incubation period and wilt severity in pepper plant; this could be as a result of inoculum threshold of the pathogen. Chatterton S, et al. [14] investigated the inoculum dose-disease response relationship for pea root- rot pathogen and reported the influence of soil type and microbial interaction on the severity of rot, especially, beyond the inoculum threshold of Aphanomyces euteiches in pea. Increase in rhizosphere concentration of phytopathogens (beyond inoculum threshold) could initiate chemical signals, leading to the expression or activation of phytoalexins against the invading pathogen [23]. This will militate against further increment in wilt severity, as observed for 107 and 108 spore/ mL E. lacerata in pepper. Although, such expression might not inhibit pathogen penetration and the consequential occurrence of wilt, it would interfere with the severity of disease expressed by the infected host.

In vitro Mycelial Inhibition of Emmia lacerata by Bacillus Strains

At 7 days after inoculation on PDA, E. lacerata FMIB29 (control) had a mycelial diameter of 66.00 mm (Table 2).

Bacillus velezensis EBs02 expressed the highest percentage mycelial control (68.33) against E. lacerata FMIB29; however, this was not significantly different from the average mycelial control (39.56%) observed for Bacillus thuringiensis EBs04. Antimycotic Bacillus species have potential to enhance crop productivity. Conventionally, Bacillus strains are screened for their ability to inhibit potential phytopathogens, usually, through the production of metabolites.

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WAI: Weeks after inoculation Mean values (± standard error). Means followed by the same letter(s) within a column are not significantly different at 5% probability level according to Duncan’s Multiple Range Test. Table 1: Pathogenicity of Emmia lacerata in pepper.

These extracellular bioactive components could serve as safer alternatives to chemical pesticides. Jan F, et al. [24] investigated the in vitro properties of Bacillus subtilis FJ3 and established the biocontrol and plant growth promoting potential of the strain. Mycelial inhibition potential of B. subtilis FJ3 was attributed to the production of hydrolytic enzymes, iturin, fengycin, biofilm and surfactin. In a similar study on the inhibitory potentials of B. velezensis, Sicuia OA, et al. [25] reported a broad spectrum of antifungal activity against phytopathogens such as Fusarium oxysporum, F. solani, F. equiseti, F. proliferatum, Penicillium expansum and strains of Aspergillus species. The antifungal potential of B. velezensis was unaffected by the features of in vitro screening (whole-cell, cell-free or volatile organic compound assay). Bacillus thuringiensis CHGP12 was also reported to produce in vitro antifungal lipopeptides and exhibited a high inhibitory potential (3.45 cm) against the mycelial growth of wilt pathogen in chickpea [26]. In addition to its in vitro properties, B. thuringiensis CHGP12 also induced a 40% reduction in wilt severity and improved the performance of infected chickpeas.

DAI: Days after inoculation Mean values (± standard error). Means followed by the same letter(s) within a column are not significantly different at 5% probability level according to Duncan’s Multiple Range Test. Table 2: In vitro mycelial inhibition of Emmia lacerata by Bacillus strains.

Effect of Bacillus Treatment on the Severity of Pepper Wilt

Bacillus strains (B. velezensis EBs02, B. thuringiensis EBs04), used as a single treatment or in combined application, significantly reduced wilt severity in infected pepper plant (Table 3). Lowest wilt severity (0.75) was observed in Emmia-inoculated plants (at 106 cfu/mL) treated with B. velezensis EBs02; however, this was not significantly higher than other inoculum treatments. Consequently, both Bacillus strains have not potentiated the wilt-biocontrol activities of one another. At the inoculum dose of 108 cfu/ mL, Bacillus EBs02 also expressed the highest wilt control, with a wilt severity score of 1.13; however, this was not significantly different from the severity scores observed for other treatments. Similar to the effect of inoculum dose on pathogenicity of E. lacerata in pepper plant, concentrations of the pathogen did not significantly influence the biocontrol potential of selected Bacillus strains. Highest wilt control (72.75%, 54.50% and 64.00%) determined for Bacillus EBs02, Bacillus EBs04 and the combination of both strains, respectively was expressed in pepper plants inoculated with the lowest concentration of E. lacerata (106 spore/mL) (Table 4). However, wilt biocontrol was not significantly different with higher inoculum concentrations (107 and 108 spore/mL) of the pathogen.

The rhizosphere of healthy plants has been established to harbor richer and more diverse microbial communities than soil associated with the root of diseased plants [27]. Consequently, biocontrol application (such as the addition of B. velezensis and B. thuringiensis) could further increase the microbial diversity in pepper rhizosphere with beneficial strains and shift rhizosphere microbial structure for a more sustainable crop production. The assembly (structure) of microbial community is determined by differential root- exudate production [28]. Rhizosphere microorganisms exist in a complex community, regulated by limiting factors such as space, nutrients and interference-competition mediated by the production of inhibitory metabolites [29]. However, exudates that selectively improve proliferation of pathogen within the rhizosphere will expose host plants to microbial attack [28]. Therefore, introduction of pathogen-suppressive bacterial species (such as antifungal B. velezensis and B. thuringiensis) could check the competitive edge accorded the pathogen by root exudation. In addition to production of bioactive antifungal metabolites, such Bacillus strains could increase microbial diversity in the rhizosphere and promote competition for space and resources (in favour of beneficial microbes).

Contrary to previous reports on improved antimicrobial efficacy associated with the combination of biocontrol agents [30], biocontrol efficacy of antimycotic Bacillus strains EBs02 and EBs04 was not additive. Combining both antifungal strains did not significantly improve wilt severity in infected pepper plants, especially, compared with the biocontrol efficacy of each strain. In a similar report, a collection of Pseudomonas strains was investigated by Vrieze M, et al. [31] for their ability to control Phytophthora infestans, the causal agent of late blight in many economically significant crops. Pseudomonas fluorescens S35 was observed to confer significant protection on three cultivars of potato when applied alone, while its biocontrol potential declined when combined with other strains of Pseudomonas species. Vrieze M, et al. [31] attributed the poor performance of P. fluorescens S35, in dual combination with other strains, to its contrasting mode of action, as well as its inability to compete for space and nutrients.