Isolation and Characterization of Wilt-Causing Pathogens of Local Growing Pepper ( Capsicum annuum L.) in Gurage Zone, Ethiopia

The yield of pepper ( Capsicum annuum L . ) is extremely threatened by diﬀerent diseases in Ethiopia. The objective of the study was isolation of wilt-causing pathogens and susceptibility test of local growing pepper. Eighteen pepper farming ﬁelds were selected for disease assessment study. The samples of Mareko Fana, Dubi, and Mitmita local cultivar pepper’s pods, seeds, leaves, stems, and roots were collected, surface sterilized, and cultured on potato dextrose agar (PDA). Selective peptone pentachloronitrobenzene (PCNB) agar medium was used for fungus. Similarly, for bacteria isolation, nutrient agar (NA) was used. Morphological and biochemical tests revealed eleven fungal isolates of Fusarium oxysporum f. sp. that were isolated. The pathogenicity test conﬁrmed nine of them were virulent to Mareko Fana, Dubi, and Mitmita local pepper. It is conﬁrmed that Fusarium oxysporum f. sp. is the pathogen Fusarium oxysporum f. sp . capsici . Besides, Ralstonia solanacearum was identiﬁed as a bacterium pathogen causing complex pepper wilt disease. The highest mean PDI was registered in Remuga Keble (93.0%) and the lowest in Buyi Keble (58.3%). Similarly, the highest mean PSI was recorded in Buyi Keble (87.0%) and the lowest PSI (54.5%) was registered in Tawlla Keble. Among 60 seeds, Mareko local pepper inoculated by F. oxysporum f. sp. and R. solanacearum shows the highest susceptibility of 55 (91.0%) and 30 (50.0%), respectively. However, Mitmita local pepper was registered as the lowest susceptibility to both F. oxysporum f. sp. and R. solanacearum of 28.3% and 30.0%, respectively . Based on the ﬁnding, it can be concluded that pepper wilt was caused by a complex of fungus Fusarium oxysporum f. sp. capsici and bacteria Ralstonia solanacearum sp. in the study area. So, it is recommended that an integrated disease management approach should be implemented to manage the complex diseases of the site.


Introduction
Hot pepper (Capsicum annuum L.) is the most important vegetable crop belonging to the family Solanaceae and grown as a spice crop in different parts of the world [1]. It is the main part in the daily consumable diet of most Ethiopian societies. Pepper production accounts for 34.0% of the total spice cultivation in Southern Nations, Nationalities, and People's Region [2]. In Ethiopia, currently, an income generating by pepper cultivation scaled up to 509.44 million Ethiopian birr for small holder farmers per year as the study conducted by Tameru et al. [3].
is indicates that hot pepper is the important source of income to farmers as an exchange earning commodity in the country.
In Southern Nations, Nationalities, and People's Region (SNNPR), Mareko, Meskan, Abeshge, Lanfro, Dallocha, Silte, Ginbo, Gibe, Gojeb, Shashego, Halaba, Meirab-Abaya, and Hawassa Zuria are high pepper-producing districts. However, the yield of the crop is low in the region. is might be attributed to the use of low-yielding varieties, drought, insect pests, and disease susceptibility [4]. Unfortunately, the total crop failure due to diseases has been common in the region and farmers are sometimes forced to abandon their production due to excessive infection pressure in the field [5].
Fungal diseases are a major risk to food security [6]. Fusarium wilt is a serious disease attacking pepper plants in Central Java which causes loss of chili yield. Fusarium wilt is caused by fungal pathogens. e symptoms of Fusarium wilt are wilting, vein clearing in younger leaflets, stunting, and yellowing of older leaves [7]. Furthermore, according to the report by Joshi et al. [8], wilt diseases caused by different fungal and bacterial pathogens are the major constraints of solanaceous crops.
As mentioned in the study by Ali [9], wilt is the main disease in pepper crops and F. oxysporum is one of the causal agents of wilting in pepper-producing countries. Fusarium isolates are the causative agent of wilt disease in a wide range of economically important crops. It is an anamorphic species circumscribed by different morphological criteria: principally, the size and shape of the macroconidia, the presence or absence of microconidia and chlamydospores, colony color, and conidiophore structure [10]. Fusarium oxysporum produces microconidia in false heads on short conidiogenous cells of monophialidic type and also produces chlamydospores, which makes it distinct. It is difficult to distinguish F. solani and F. subglutinans. However, F. solani forms microconidia in false heads on very long monophialidic conidiogenous cells. F. subglutinans is distinguished from F. oxysporum by the formation of microconidia from mono-and polyphialidic conidiogenous cells and lack of chlamydospores [11].
In this study, Abeshige, Mareko, Meskan, and Kebena districts are potential areas for pepper crops [4]. However, yield is highly affected by pepper wilt-causing pathogens, which leads to total loss of productivity. Type and relative importance of each disease across locations have not been well characterized and profiled in the study area. So, it is important to isolate and identify types of pathogens for disease control and management in the area. e objective of the study was isolation and characterization of wilt-causing pathogens and analyzing susceptibility of local growing pepper (Capsicum annuum L.).

Description of the Study Areas.
e study was conducted in three districts of Ethiopia: Gurage Zone of Southern Nations, Nationalities, and People's Region (SNNPR). Abeshige district (Lache-omancho and Dalga Keble), Kebena district (Remuga and Qola-kabada Keble), and Mareko district (Enseno and Buyi Keble) were selected to represent the major pepper-growing areas of the zone. Experimental activity was conducted in the Biotechnology Department laboratory of Wolkite University. Wolkite town is located 171 km away from Addis Ababa, the capital city of Ethiopia. e districts have average latitude and longitude of 8 o 17′N and 37 o 4′E and an elevation between 1537 and 2010 m above sea level.

Field Assessment and Sampling Techniques.
ree potentially pepper-growing districts, namely, Abeshige, Kebena, and Mareko, of Gurage Zone were selected. From each district, six pepper farming fields were selected. Farm fields at each selected area were systematically sampled to collect disease specimens. Totally, 100 pepper plant samples were collected for disease measurements from 3 m × 2 m quadrant in each farm field. During this study, 100 peppers from each field, that is, 1800 from eighteen fields, were assessed for percent disease incidence (PDI) and percent severity index (PSI) determination in the 2019 cropping season. Pepper wilt was observed in all surveyed localities, with varied disease intensity.

Isolation and Characterization of Wilt-Causing
Pathogens. Pepper leaves, stems, and roots were collected from wilt disease symptom-showing peppers, kept separately into polythene bags, placed inside the ice box, and brought to the Biotechnology Laboratory of Wolkite University. Pepper plant leaves, stems, and roots were cut into smaller pieces, and seeds were surface sterilized in 70% alcohol and 0.5% sodium hypochlorite for 30 and 60 s, respectively. en, they were rinsed in sterile distilled water and transferred to Petri plates. For isolation of different fungal and Fusarium fungi pathogens, potato dextrose agar (PDA) and selective peptone pentachloronitrobenzene (PCNB) agar medium were used. e peptone PCNB agar medium ingredients were 15 g Difco peptone, 1 g KH 2 PO 4 , 0.5 g MgSO 4 ·7H 2 O, 20 g agar, 1 g pentachloronitrobenzene (PCNB, 75% WP), 1 mL lactic acid, 0.5 g chloramphenicol, and 1 L distilled water. e ingredients were mixed and dissolved, the pH was adjusted to 4.5, and then the medium was autoclaved [12]. e Petri plates were incubated at 28°C for 4 days followed by colony morphology identification. Four-day grown fungal hyphae were further subcultured on the potato dextrose agar (PDA) medium amended with 300 mg/L chloramphenicol. A colony of fungus with different colors on PDA near the pepper sample parts was selected and transferred into new PDA and incubated for 7 days to get pure culture of the isolate. For bacterial isolation, nutrient agar was used.

Morphological Examination of Isolated Fungus.
After pure colony development, identification of fungi was performed based on the cultural characteristics and microscopic examination using the standard manuals [13]. Colony color, number of septation, and the shapes of spore (macroconidia and microconidia) were recorded and used for morphological classification. e spores of fungi were taken aseptically, mounted on a slide, stained with methyl blue, and covered with a cover slip to examine under a microscope (100x). Identification was made on the basis of their colony morphology (shape and color), spore shape, shape and size of microconidia, size and septation of macroconidia, presence of isolate, or chain chlamidospores. Morphology results were compared using text books for identification of fungi of their taxonomic keys, as stated by Ronhede et al. and Ulhan et al. [10,14]. e identity of the culture was further confirmed only with the presence of macroconidia and microconidia. Pure cultures of all the isolates were stored on PDA [15].

Morphological and Biochemical Characterization of Isolated Bacteria.
e pure colonies were subjected to various morphological and biochemical characterization to determine the isolated bacteria according to Sonkar et al. [16]. e performed morphological tests were Gram staining and tests for color and shape of colony. e performed biochemical tests were the catalase test, oxidase test, citrate utilization test, starch hydrolysis test, KOH test, and urease tests. e bacterial streaming test was applied as a method to distinguish bacterial wilt. e infected plant stem was cut and dipped into a water-containing test tube. e bacterial exude from the cut end was analyzed [6].

Disease Incidence Assessment.
is was made by observing wilting and leaf-yellowing expression symptoms on representative plants to determine the general presence or absence of diseases [17]. Pepper-farming fields were visited diagonally, and the disease incidence was estimated by using the 3 m × 2 m quadrant. e percent disease incidence was calculated by using the following formula: number of plants affected total number of plants observed × 100. (1) To know disease severity, symptoms such as wilting, vascular discoloration, and root rotting were considered as indices for sample collection. Root rot and wilt severity were estimated from 10 days to 35 days by 5 days of interval after transplanting by following the method outlined by Ismail et al. [11]. e disease rating scale of 0-5 was based on leaf yellowing and wilting grading: 0 � healthy, 1 � one leaf yellowing, 2 � more than one leaf yellowing, 3 � one wilted leaf, 4 � more than one leaf wilted, and 5 � completely dead/ wilted plants.
Disease severity scores were converted into percentage severity index (PSI) as follows: sum of numerical rate number of plants scored × maximum score on scale × 100. (2)

Pathogenicity and Susceptibility Test.
ree different genotypes of local peppers, Mareko Fana, Dubi, and Mitmita, were used for both R. solanacearum and Fusarium oxysporum pathogenicity tests. A total of 60 seeds were selected for each cultivar and then surface sterilized and planted on a plastic pot with 25 cm diameter containing autoclaved top soil, composite, and sand at 3 : 2 : 1 ratio, respectively, according to Fekadu and Dandena [4]. Each pot was made to have 12 seeds with three replications for each local pepper cultivar. e experiment was carried out under greenhouse condition at 25°C. A completely randomized design was used with three replicates (pots). A healthy plant was maintained as the control. Percent disease incidence and disease severity were recorded. e pathogenicity test for R. solanacearum was carried out according to the method outlined by Mimura and Yoshikawa [18]. e bacterium was cultured in nutrient broth media overnight on an incubator shaker, and the concentration was adjusted using a UV spectrophotometer. Soil on plastic pots was infested with 5 mL of (4 × 10 8 cfu/ mL) bacterial suspension into the center. After thirty days, three pepper seedlings were transplanted into a R. solanacearum inoculated pot with 25 cm diameter under greenhouse condition. e inoculated pepper plants were examined for wilting symptoms starting from 10 DAI (days after inoculation). Disease symptom and proportion of wilted plants (PW) were recorded by 5 days of intervals till 35 DAI. e pathogenicity test for isolated Fusarium sp. was performed using the method designed by Khalifa [19]. Five milliliters of conidial suspensions containing 6.4 × 10 6 conidia/mL were used to infest the soil on pots.
irty-day-old three local peppers used in this study were transplanted onto the infected soil in pots with three replications. Sets of control pepper from all genotypes were subjected to sterile soil-containing pots. Pepper wilting symptom examination and proportion of wilted plants were registered starting from 10 DAI every 5-day interval till 35 days as stated for R. solanacearum. For further confirmation test, isolates identified using cultural or morphological characteristics and yellowing lesions on the vascular bundle were examined. e pathogen isolated in the laboratory was compared with that isolated from disease symptom-developing pepper. Disease susceptibility was calculated, and grading was performed on the basis of wilt characteristics which is expressed by percent as follows: highly resistant (HR), 0% wilting; resistant (R), 1-10% wilting; moderately resistant (MR), 11-20% wilting; moderately susceptible (MS), 21-30% wilting; susceptible (S), 31-50% wilting; and highly susceptible, >50% wilting, according to Bayoumi and El-Bramawy [20].

Results and Discussion
A total of eleven fungal colonies (PF1-PF11) and two bacterial colonies (BC1 and BC 2) were isolated from pepper leaves, seeds, stems, roots, and pods. From the isolated fungi, nine isolates were found to be pathogenic to Mareko Fana, Dubi local, and Mitmita local pepper plant showing typical wilt symptoms on the foliage growth, stems, and xylem vesicles. Similarly, bacteria isolates were found to be virulent to these three selected genotypes of local pepper plants.

Isolated and Identified Types of Pepper Wilt-Causing Fungi
Pathogens. Table 1 shows that all colonies grown on the plate showed light brown pigmentation in PDA except PF-10 and PF-11 isolates. is result was similar to that reported by Ismail et al. and Sonkar et al. [11,16]. e fungal isolates coded as PF-3, PF-4, PF-6, and PF-9 registered the largest colony with averages between 5.3 μm and 5.5 μm diameter.

International Journal of Agronomy
Isolates such as PF-1, PF-2, PF-5, PF-7, and PF-8 formed colonies that cover a small part of the plate on PDA media with white mycelia and red-brown pigmentation, which was the same as observed in the study reported by Balali and Iranpoor [21]. On the basis of mycelium growth up pattern, all isolates showed adherent smooth surface except PF-10 and PF-11 with fluffy growth on PDA.
All isolates' microconidia were devoid of septation and macroconidia were with septation ( Figure 1). Microconidia were found singly or in mass with cylindrical shape. e isolates in this study registered nonseptate microconidia formed in false heads on short monophialides. Septation of macroconidia were found to be 2, 3, and 4 on PF-1, PF-3, and PF-9; PF-4, PF-5, and PF-6; and PF-2 and PF-7, respectively. Macroconidia found in this study were the same related to the shape falciform, number of septation (2)(3)(4), and abundance as the finding by Ciampi et al. [22]. For Fusarium oxysporum sp., F. solani forms cylindrical macroconidia, with no convex curvature, but F. oxysporum f. sp form falciform/ellipsoid shape according to Ulhan et al. [10]. Oval-shaped microconidia without septation and falciformshaped macroconidia possessing 2-4 septation were registered. All fungal isolates were identified to have short phialides. Cultural and biochemical analysis of the bacterial study showed that R. solanacearum was found as a wiltcausing pathogen on peppers.
All fungal isolates except PF-9 and PF-2 were identified to contain short phialides of less than 18 µm. is characteristic made our isolate, F. oxysporum, different from F. solani that contains long phialides. It is the best characteristic for differentiating these two species according to Balali and Iranpoor [21]. So, in concert with colony characteristics and microscopic structures, all the nine fungal isolates showed the characteristics of F. oxysporum f. sp. In this regard, the result was in line with the work of Pawasker et al. [23]. e average macroconidia length and width ranged from 5.5 × 3 μm to 10 × 3 μm. As indicated in this study, chlamydospores existed in single and intercalary in PDA cultures except on PF-2 and PF-9 isolates.

Morphological and Biochemical Characterization of Isolated Bacteria.
One of the causative agents of pepper wilt disease identified in this study was a bacterial pathogen. As morphological and biochemical tests revealed, two bacterial colonies (BC1 and BC 2) were identified as Ralstonia solanacearum (Table 2). e biochemical test result was similar to the work of Assefa et al. and Pawasker et al. [6,23]. is bacterial isolate was found infecting the pepper alone and with the association of F. oxysporum f. sp.

Disease Measurement.
Mean percent disease severity (PSI) and percent disease incidence rate (PDI) were found to be 65.9% and 73.2%, respectively (Table 3).
Percent severity index and percent disease incidence of pepper wilt disease within the selected kebles are shown in Figure 2. is study showed that, in three pepper-farming districts, the highest mean disease incidence (PDI) was registered in Remuga Keble (93.0%) and the lowest was observed in Buyi Keble (58.3%). e highest mean PSI was also recorded in Remuga Keble (87.0%), and the lowest PSI (54.5%) was registered in Tawlla Keble. e mean PSI (65.9) showed high severity of disease in the area according to the disease severity rating set by Bayoumi TY [20]. During the study period, the mean PDI was found to be 73.2% which is higher than the result reported by Shiferaw and Alemayehu [24] in Abeshige district. e PSI of the study area was higher than the result reported by Mekonnen et al. [6], a research conducted in Bako Tibe district, Oromia Regional State. e occurrence of high PSI of pepper wilt disease in the area may be owing to the high soil-water holding capacity, cultural practices, type of the cultivars used plant spacing, and level of inoculums present in the soil [25].
Both the fungal isolate F. oxysporum and the bacteria isolate R. solanacearum were found from stems, roots, leaves, pods, and seeds of infected pepper plants. Both the isolates were found together forming a complex pepper wilt disease-causing pathogen [24]. is study shows the reason for limited productivity and total loss of pepper yield through identification of the main causative agent of pepper wilt disease. Also, Temam [26] reported Fusarium wilt (Fusarium oxysporum) as being the most widespread fungal disease of hot pepper in Ethiopia. e field observation symptoms of wilt-causing diseases were wilting, vein clearing in younger leaflets, stunting, and yellowing of older leaves (Figure 3). e pathogenicity test of F. oxysporum isolates revealed that the nine tested isolates were pathogenic to Mareko Fana, Dubi local, and Mitmita local pepper plants showing typical wilt symptoms on the foliage growth, stems, and xylem vesicles. Moreover, R. solanacearum bacteria inoculated onto local growing pepper showed symptom spreading from the youngest leaves to the oldest leaves which made it different from the fungus F. oxysporum which is in line with the description given by Lowell et al. [17]. e wilting characteristics due to F. oxysporum started from the older leaves and spread to the younger ones. A transverse cut of stem also showed vascular browning that confirms most likely F. oxysporum and bacterial ooze for R. solanacearum. Roots and stems of the diseased pepper plant that were cut and placed in a small test tube containing water showed yellowish ooze coming from the cut end. is ooze was a key feature in confirming the disease caused by bacterial pathogens. is finding was similar to the work of Chaudhuri et al. [27].

Conclusion
Cultural, microscopic analyses and biochemical tests supported by the pathogenicity test revealed that fungi Fusarium oxysporum capsici and bacteria R. solanacearum were the causative agents for pepper wilt disease in the study area. From the findings, local growing pepper, Mareko Fana, and Dubi local pepper were identified highly susceptible to Fusarium oxysporum capsici and R. solanacearum, but Mitmita local was identified moderately susceptible to both pathogens. e current study indicated that a complex of diseases exists at each growth stage of pepper and the occurrence across districts is highly variable. erefore, an integrated approach is required to manage the complex diseases in the area. Also, pepper cultivars should be selected, tested, and genetically improved for the wilt-causing pathogen control.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.