The Biocontrol of Soil Transmitted Cercospora Capsici with Lactobacillus Plantarum

Journal of Advances in Microbiology

18(3): 1-8, 2019; Article no.JAMB.51763 ISSN: 2456-7116

The Biocontrol of Soil Transmitted Cercospora capsici with Lactobacillus plantarum

Oluwafemi M. Adedire1*, Ayotunde Pitan2, Adekunle O. Farinu1 and Wuraola F. Ogundipe1

1Federal College of Agriculture, P.M.B. 5029, Apata, Ibadan, Nigeria. 2National Horticultural Research Institute, P.M.B. 5432, Idi-ishin, Ibadan, Nigeria.

Authors’ contributions

This work was carried out in collaboration among all authors. Authors WFO and AOF contributed to the in vivo biocontrol assay and reviewed the first draft of the article. Author OMA established the experimental design and coordinated the microbial isolation procedure. Author AP reviewed the draft of the article and contributed to the publication process. All authors read and approved the final manuscript.

Article Information

DOI: 10.9734/JAMB/2019/v18i330173 Editor(s): (1) Dr. Pradeep S. University of Calicut, Kerala, Jawaharlal Nehru Tropical Botanic Garden & Research Institute,Kerala, India. Reviewers: (1) Karim Houali, Mouloud Mammeri University of Tizi-Ouzou, Algeria. (2) R. Mahalakahmi, India. Complete Peer review History: http://www.sdiarticle4.com/review-history/51763

Received 01 August 2019 Original Research Article Accepted 04 October 2019 Published 21 October 2019

ABSTRACT

Microbial diseases of pepper (Capsicum species) are the most significant factors contributing to the loss of this economically important vegetable crop. Leaf spot disease caused by Cercospora capsici is one of the major constraints to pepper production in Nigeria. The disease is reportedly introduced through infected seeds. However, the development of Cercospora leaf spot on plants propagated from fungicide-treated seeds suggests an alternative perennial source of infection and also necessitates the development of an efficient, safe control measure. C. capsici was isolated from infected pepper plant through the direct plating method and subsequently characterised. Treated pepper seeds (with L. plantarum) were planted in C. capsici inoculated soil, while the emergence, seedling growth parameters and severity of leaf spot were observed. The severity index of Cercospora leaf spot on pepper plants (observed at the 20th day after planting) was significantly lower on L. plantarum treated plant set (0.07) than on pepper without seed treatment. Seed emergence rate index increased from 11.11 to 15.33% /day of untreated to treated pepper seeds sown in infected soil respectively, while the mean emergence time of untreated seeds (8.32 days) ______

*Corresponding author: E-mail: [email protected];

Adedire et al.; JAMB, 18(3): 1-8, 2019; Article no.JAMB.51763

was significantly higher in C. capsici infected soil. It could therefore be deduced that seed priming with L. plantarum improved the seedling vigor and resistance of pepper to leaf spot disease caused by C. capsici.

Keywords: Pepper; Cercospora capsici; Lactobacillus plantarum; biocontrol.

1. INTRODUCTION necrotic and sometimes accompanied with leaf chlorosis. Although, C. capsici is mostly Capsicum chinense Jacq. belongs to the family introduced through infected pepper seeds, the Solanaceae, it is one of the important cash emergence of Cercospora leaf spots on pepper earning crops globally, mainly cultivated as a fruit plants grown from treated seeds suggests other vegetable [1]. It is a commercially important sources of primary infection. It is therefore vegetable crop in Nigeria [2], grown in almost all imperative to investigate the potentials of soil as the States under rainfed or irrigated conditions a reservoir of inoculum for pepper, considering for both domestic and export (through the the possibilities of infected crop residues (within Nigerian Export Promotion Council) purposes. the soil) as a platform of seasonal pathogen Nigeria accounts for about 50% of African pepper carryover. production, making Nigeria the largest producer of pepper in tropical Africa [3]. Chilli pepper has The use of chemical fungicides to control been described as an increasingly important microbial diseases of plants leads to the vegetable crop. It is used for culinary purposes accumulation of toxic chemicals, which could as spice and food colorant; as well as for pose a potential threat to human health upon medicinal applications. The consumption of Chilli consumption and their non-selective toxicity pepper has, over the last 35 years, increased could negatively alter the balance of soil beyond 40-folds [4]. This economically significant beneficial microorganisms [8]. However, Lactic vegetable crop is an important source of Acid Bacteria (LAB), especially, Lactobacillus essential food nutrients, including vitamins K, C plantarum, had been documented to inhibit and B, lycopene (carotenoid), calcium and trace several animal and plant pathogens in vivo and metals, which could provide a long term health in vitro without generating undesirable effects on benefit to consumers [5]. Natural, bioactive, the host [9]. The alternative use of these pharmacological compounds associated with antagonistic Generally Regarded as Safe pepper include the phytochemicals, capsaicin, (GRAS) microorganisms to control essential oils, a rich combination of organic phytopathogens of pepper would improve the micronutrients and Reactive Oxygen Species survival of pepper, reduce the yield loss currently (ROS)-protective antioxidants [6]. associated with this vegetable crop and improve its production in Nigeria. Consequently, this The production of pepper in Nigeria is limited by research is designed to investigate the control several factors including diseases caused by effect of Lactobacillus plantarum on Cercospora fungi, bacteria, viruses and nematodes, as well leaf spot disease of pepper. as physiological imbalances caused by abiotic stress. Among the microbial diseases 2. MATERIALS AND METHODS responsible for pepper yield loss worldwide, fungal diseases are the most frequent [1]. Leaf 2.1 Sample Collection and Experimental spot caused by Cercospora capsici Heald and Sites Wolf is a major constraint to pepper production in Nigeria, causing foliar pathogen damage with Infected plant samples showing symptoms of epidemic potentials, due to its exponential Cercospora leaf spots were collected from spread among susceptible varieties [7]. different farms at the Ibadan/Ibarapa agricultural Cercospora leaf spot usually begins to manifest zone, Oyo State, Nigeria (7°23'39.6"N; at the base or lower surface of infected leaves, 3°47'02.9"E /7°26'14.5"N; 3°16'3.4"E). Samples where there is more moisture and less wind or air were collected in bags, transported to the circulation and subsequently spreads outwards. laboratory and kept in the refrigerator at 4ºC The symptoms on leaves appear as circular (up (overnight) when isolation could not be done to 1 cm diameter), small, brown to black spots immediately. Laboratory analyses were carried with a small (0.5–1 mm) whitish centre that out at the Microbiology Laboratory of the Federal slowly enlarges, which could become severely College of Animal Health and Production, while

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the pot experiment was done at the Horticulture early emergence and overall performance, were unit, Federal College of Agriculture, Moor surface sterilised with 1% NaOCl for two Plantation, Ibadan (7°22'48.5"N; 3°50'29.0"E). minutes, drained on sterile filter paper and planted 24 hours after soil inoculation. The 2.2 Isolation of C. capsici from Infected manifestation of leaf spots was recorded and Pepper Plants compared. The most virulent strain was re- characterised (identified) and kept in the Using a sterile scalpel, 5 mm portion of infected refrigerator at 4ºC for subsequent analyses. pepper leaf was cut from each plant sample. The separated plant tissues were surfaced sterilized 2.5 In vivo Control of Cercospora Leaf with 1% sodium hypochlorite (NaOCl) solution for Spot with L. plantarum 2 minutes. Treated samples were subsequently rinsed thrice in sterile distilled water (SDW), 2.5.1 Seed treatment drained on sterile filter paper and inoculated on solidified, presterilised (121ºC at 15 psi for 15 Soil inoculation was done as earlier described in minutes) Potato Dextrose Agar (PDA) using the section 3.4 and seed treatment with LAB species direct plating method. Three replicates were was carried out by biopriming surface sterilised 6 made for each sample and the inoculated Petri seeds with 10 CFU/mL purified L. plantarum plates were incubated at ambient temperature (Earth's pearl™). Treated seeds were planted in (25±2) for 5-7 days. Fungal growth on each plate inoculated soil (15 mm depth) at the rate of 8 was then subcultured on fresh PDA plates to seeds per pot [12]. Negative control seeds and obtain pure cultures of each strain [2]. soil were treated with SDW (treatment-C), while the soil of treatment-A plant set was inoculated 2.3 Identification of Isolated Fungi with C. capsici without seed treatment. Both the soil and the seeds of treatment-B plants were The purified fungal isolates were identified by inoculated with the pathogen and Lactobacillus examining them macroscopically and species respectively. The seeds of treatment D microscopically. Colony properties such as the plant set were bioprimed with L. plantarum appearance, change in medium colour and without soil treatment. Emergence was scored growth rate were observed. Microscopic when the hypocotyl hook was seen on the soil structures like the shape of hyphae, conidia and surface, while emergence parameters were conidiophores were observed. These structural measured and seedling properties were features were compared with standards [10]. recorded.

2.4 Pathogenicity Test 2.5.2 Measurement of control and biostimulating effects of L. plantarum Pathogenicity test was carried out to investigate The severity scale of 1-5, as described by Asare- the aggressiveness of the isolates and measure Bediako et al. [13], was used to investigate the their virulence on each infected plant. Fungal control effect of seed treatment and the scoring suspension was prepared for soil inoculation. was translated to disease severity index (DSI). This was done by subculturing (on PDA) pure isolates from the edge of an actively growing % Disease severity index (DSI) = plate and incubating at room temperature for 5 days. Sterile distilled water (30 mL) was ∑( × ) × 100 [14] thereafter poured over the surface of the fungal × growth and a sterile spatula was used to scrub the surface of the fungus to dislodge the Seeding dry weight (SDW) and seeding fresh mycelium over a sterile cheesecloth filter to weight (SFW) were determined, while collect the fungal spores (filtrate). emergence percentage, emergence rate index Hemocytometer was used to adjust the spore [15], seed vigor index, mean emergence time concentration of the fungal suspension to 105 [16] and disease incidence [11] were measured conidia/mL. Presterilized sandy loam soil (5 as described below: kg/pot) was then inoculated with 25 mL of the prepared fungal suspension a day prior to i. Emergence percentage planting and the pot was kept humid with SDW [11]. Seeds of Capsicum chinense Jacq. (rodo- Number of emerged seeds Emergence (%) = × 100 wewe), a variety commonly cultivated due to its Total number of seeds

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ii. Emergence rate index (ERI) 3.2 Pathogenicity Test

Emergence rate index (%/day) The development and spread of leaf spots by C. capsici from the lower to upper surface of the leaf 5% 10% 15% 35% (Fig. 1), as well as from the older pepper leaves = + + + ⋯ 5 10 15 35 to the younger ones could probably be due to the amount or types of nutrients, structures and Where Gx% is the percentage emergence at day moisture content present on these leaves. As the x after planting disease progresses, infected plants that do not collapse or die off from infection have been iii. Seed vigor index (SVI) reported to produce lower fruit yield [7]. Leaf spots, spreading from the lower surface to the Seed vigor index = Seedling length × G35% upper surface of the leaves, appeared as reddish brown spots which later turned to grey spots. Where G35% is the percentage emergence at Infected areas formed circular necrotic lesions, day 35 after planting most times accompanied with chlorosis, which eventually led to wilting. Aggressiveness of C. iv. Mean emergence time (MET) capsici has been attributed to the ability of most pathogenic strains to produce cercosporin, a Mean emergence time (day) phytotoxic polyketide metabolite. The ability to produce cercosporin, as well as other virulence ∑(Emergence Number × day) metabolites like enzymes and host specific toxins = ∑(Emergence Number) could significantly enhance virulence in C. capsici and aid the manifestation of spots v. Disease incidence through tissue disruption and depolymerization [17,18]. Number of infected plants % Incidence = × 100 3.3 In vivo Biocontrol of C. capsici Total number of plants As reported by Islam et al. [19] in their 2.6 Statistical Analysis investigation on the diagnosis and prescription for Cercospora leaf spot of chilli (using the plant The recorded data were subjected to one-way disease diagnosis model), they proposed analysis of variance (ANOVA) with the aid of strategies in the control of this disease. They Statistical Package for the Social Sciences observed significant increment in the severity of (SPSS) software adapted for Windows, version untreated pepper plants at 80 and 95 days after 16.0. Each treatment was replicated four times in planting. This affected the growth parameters, a Completely Randomized Design (CRD). weight per fruit and the number of fruits per Analysed means were separated with the plants. The incidence of Cercospora leaf spot (as Duncan Multiple Range Test (DMRT at P≤0.05). observed in this study) was reduced by 7.34, 1.14, 0.5 and 1% at the 5th, 10th, 15th and 20th 3. RESULTS AND DISCUSSION days after planting respectively (Table 1) through pre-planting seed biotreatment with cell 3.1 Identification of C. capsici suspension of L. plantarum. The disease severity index of Cercospora leaf spot was significantly The isolate was observed to form a light brown reduced in L. plantarum treated plants, compared mycelial growth upon macroscopic morphological to plants grown in infected soil without seed examination. The growth was rapid and irregular, treatment. Highest reduction in the disease with patches (2-3 cm) of mycelia forming 3-4 severity index (1.0) was observed at the 20th day days after inoculation. Microscopic examination after planting. revealed acicular conidia (55-140 x 3-5 µm), hyaline hypha, septate (5-8) and smooth. The Similar biocontrol results were observed by hyphae are either curved or straight with round Devappa and Thejakumar [1], who treated C. tips and basal truncated cells with characteristic capsici infected pepper plants with lyophilized scars. The macroscopic, microscopic and culture of Pseudomonas fluorescens as an in disease characteristics were compared to vivo control measure. Disease index (DI) from standards and the fungal isolate was identified as their study was recorded at 45, 60 and 75 days C. capsici. after transplanting; they reported a percentage DI

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A B C

Fig. 1. Pathogenesis of cercospora leaf spot. A: Development of spots at the lower surface of leaf. B: Spread of spots to the upper leaf surfaces. C: Wilting and stem collapse of pepper plant range of 8.00 to 21.33 for treated plants, as height of the plant. Taking cognizance of the compared to the control plants (22.33 - 45.33%). primed and unprimed seeds planted in Abdel-Aziz et al. [20] also recorded a significant uninfected soil, the improvement in performance reduction in the severity of fungal diseases on of treated pepper seedlings observed in this plants grown from seeds bioprimed with Lactic study could be as a result of direct production of Acid Bacterial species. They demonstrated the plant growth stimulating metabolites by L. capability of LAB species as effective biocontrol plantarum, in addition to direct inhibition or the agent against phytopathogenic fungi through in competitive exclusion of leaf spot pathogen (C. vitro and in vivo assays. Strains of L. plantarum capsici). Samarah et al. [15] equally described have been described to produce a complex the growth promoting and protective properties of combination of antimicrobial compounds. These glucosamine polymers on treated pepper seeds. compounds include antimicrobial proteins Nanochitin and chitosan are natural biopolymers (bacteriocins), diacetyl, hydrogen peroxide and of arthropod or microbial origin with organic acids with broad to narrow spectrum of biostimulating potentials (including improved inhibitory activities [21]. Comparative genomic seedling growth and enhanced seed germination analysis of Lactobacillus plantarum had been properties). These long-chain polysaccharide used to investigate over 1425 protein-coding structures could also induce host defense genes (within the core genome of this species), mechanisms through tissue fortification some of which are involved in antibiosis [22]. (lignification), expression of enzymes, production Sodium dodecyl sulfate–polyacrylamide gel of reactive oxygen species and activation of electrophoresis of partially purified antimicrobial primary, as well as systemic defense protein from L. plantarum revealed a molecular mechanisms. weight range between 12 and 45 kDa [21]. 3.5 Effect of Seed Treatment on the 3.4 Effect of Seed Treatment on the Emergence Parameters of Pepper Growth of Pepper Seedlings Seedlings

Seed treatment with L. plantarum also improved Seed treatment appeared to improve the rate of the emergence of pepper seeds used in this emergence (emergence rate index), the average study (Table 1). Seeds planted in infected soil emergence period (mean emergence time), without LAB treatment had the lowest % seedling dry weight and the seedling vigor index th emergence (33.33) at the 5 day after planting. of pepper (Table 2). Infected pepper seedling Bioprimed seeds planted in uninfected soil had without L. plantarum treatment had the lowest the highest number of leaves at 10, 15 and 20 seedling dry weight (0.07 g), mean emergence days after planting. However, biopriming pepper time (8.32 days), emergence rate index (11.11 seeds did not significantly influence the seedling %/day) and seedling vigor index (860.00). L.

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plantarum seed treatment significantly Similar biostimulating results have been reported biostimulated pepper seeds with a seed vigor on other nightshade crops. Tomato seeds primed index value of 1.350 × 103. In a similar study, the with Streptomyces fradiae NKZ-259 suspension biopriming of wheat seeds with cell suspensions improved seed emergence and seedling vigor of L. plantarum ONU12, L. plantarum ONU311 through the production of Indole-3-acetic acid and L. plantarum ONU355 strains was reported [24]. Mavi [25] also evaluated the effect of to increase seed germination by 6.0 to 40.0 % organic priming on the emergence performance depending on the substrate used and the of five domesticated Capsicum species (C. inoculum concentration. Limanska et al. [23] frutescens, C. baccatum, C. annuum, C. reported an optimum inoculum concentration chinense and C. pubescens). The author range of 103 to 106 CFU/mL for wheat seed reported improved emergence vigor index, higher biotreatment under hydroponics and soil seedling length values, as well as higher conditions. L. plantarum was observed to form fresh and dry seedling weights of Capsicum biofilms on roots of seedlings and improved the species primed with aqueous extract of Tagetes height, as well the root length of host plants [23]. patula L.

Table 1. Protective and biostimulating effects of L. plantarum on pepper

Days after planting Treatment % Emergence Seedling Leaf Incidence Disease (DAP) height number (%) severity index 5 DAP A 33.33b 3.37 2.33ab 20.17a 0.11a B 63.33a 3.92 2.83ab 12.83a 0.04b C 56.67a 3.40 2.03b 0.00b 0.00b D 60.00a 4.85 3.10a 0.00b 0.00b Sig. * ns * * * 10 DAP A 40.00a 4.27 4.03b 2.97a 0.14a B 20.00b 4.34 4.40ab 1.83ab 0.06b C 23.33ab 5.08 4.67ab 0.00b 0.00c D 20.00b 5.87 5.10a 0.00b 0.00c Sig. * ns * * * 15 DAP A 6.67 6.06 5.37ab 2.50a 0.16a B 10.00 6.67 5.30ab 2.00a 0.07b C 6.67 7.20 5.09b 0.00b 0.00c D 10.00 7.57 5.97a 0.00b 0.00c Sig. ns ns * * * 20 DAP A 0.00 6.18 5.57 2.50a 0.17a B 0.00 6.80 6.00 1.50b 0.07b C 0.00 7.38 6.17 0.00c 0.00c D 0.00 7.83 6.13 0.00c 0.00c Sig. ns ns ns * * ns: not significant; *: Significant; Mean values with similar letter (s) along the column are not significantly different at 5 % level of probability by Duncan Multiple Range Test (DMRT)

Table 2. Effect of seed treatment with L. plantarum on the emergence and performance of pepper

Treatment SFW (g) SDW (g) MET (day) ERI SVI (× 103) A 1.37b 0.07b 8.32a 11.11b 0.860c B 2.13ab 0.20a 7.17b 15.33a 1.161b C 2.23ab 0.23a 7.10b 13.60a 1.184b D 2.33a 0.27a 7.21b 15.17a 1.350a Mean values with similar letter (s) along the column are not significantly different at 5 % level of probability by Duncan Multiple Range Test (DMRT)

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Peer-review history: The peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/51763