PATHOGENICITY OF CONIELLA MUSAIENSIS VAR. HIBISCI, A CAUSAL AGENT OF ROSELLE SPOT DISEASE IN MAKURDI, CENTRAL

Kparev-wua David APEYUAN1; Okechukwu Alphonso NWANKITI2; Apeh Ocheigwu Hyacinth OLUMA3, Ebenizer Jonathan EKEFAN2 1Department of Integrated Science,College of Education, Katsina-Ala, Benue State. 2Department of Crop and Environmental Protection University of Agriculture, Makurdi, Benue State. 3Department of Biological Sciences, University of Agriculture, Makurdi, Benue State. *Correspondent Author: [email protected]

Abstract

A leaf spot disease was observed in Benue State, Nigeria in the 2013 cropping season. of infected appeared water-soaked. These are usually small, irregular and light brown. These spots increased in size covering the leaf surface and bleaching it. These spots eventually become darkened, necrotic and distorted. Affected leaves last for only three days and dropped off. The pathogen was isolated from infected leaves, seeds, crop debris and soil on potato dextrose agar supplemented with streptomycin sulphate (PDAS). The pathogen was identified as Coniella musaiensis var. hibisci by the Global Clinic in the United Kingdom (GPCW No. /w 9181). Symptoms were reproduced 24 hours after artificially inoculating wounded leaves of the accessions (Acc1, Acc3 & Acc4) tested. No significant (p=0.05) difference was noticed among the accessions with regards to disease severity. The infected leaves were defoliated after 72 hours. It was noticed that the disease did not spread to newly emerged leaves after the dropping of the infected ones. Progression ceases once there is low humidity. This fungus has been previously reported elsewhere in the country but this is its first report in Benue State, Nigeria

Keywords: leaf spot, Coniella musaiensis var. hibisci, sabdariffa L., accession, inoculation.

Introduction

Roselle (Hibiscus sabdariffa L.) is an important vegetable crop in Nigeria. It belongs to the family . More than 300 accessions of it can be found now around the world, growing in both tropical and sub-tropical regions. Most accessions are used as ornamental plants, but the swollen red calyces of the sabdariffa type have economic, nutritional and medicinal values (Tindall, 1983; Morton, 1987; Sharoff, 1996; Onyenekwe, 1998; Alegbejo, 2000 and Qi et al., 2005). According to Schippers 2000, Roselle is ranked the third most important vegetable in most Nigerian markets, coming after tomatoes and okra in that order. Nigeria is not listed among producing nations which is an indication that the production in the country has not been placed on international scale. Nevertheless, meaningful and substantial production has been reported in the country mainly in the Guinea and savanna zones with major growing areas being Niger, Kogi, Oyo, Kaduna, Bauchi and Kwara States (Alegbejo, 2000). The crop has not yet attained its full economic potentials in the country yet there are challenges militating against its optimal growth and development. This leaf disease caused by Coniella musaiensis var. hibisci is a major constraint (Alegbejo, 2000) and management of the disease has to start with ascertaining the pathogenicity of the causal agent. Therefore the aim of the study was to establish the identity and ascertain the pathogenicity of the causal agent.

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Materials and Methods

Isolation of the Coniella musaiensis var. hibisci The pathogen was isolated from infected leaves in the 2013 cropping season using the method described by Agrios (2005) in the advance pathology laboratory of University of Agriculture Makurdi, Nigeria. Infected leaves were collected from the field at four locations, placed in envelopes and carried to the laboratory. The infected leaves were thoroughly washed in distilled water. Several small sections (5mm2) were cut, using sterile scissors, from the margin of the infected lesions to contain both diseased and healthy portions of the tissue. The sections were surface sterilized with a dilution of common household bleach (0.5 % active sodium hypochlorite) at the ratio of 1:10 for 1 minute and blotted dry on clean sterile filter papers. The sections were transferred to Petri dishes (4 per dish) containing potato dextrose agar supplemented with 0.1mg/l streptomycin sulphate (PDAS) using sterile forceps. The plates were incubated for seven days at room temperature. Colonies of the organism were sub- cultured by using a sterile needle to remove small portions of the growth and transferring them to fresh PDAS plates, a method described by Collins and Lyne (2004). Subsequent sub- culturing was done until pure cultures were obtained. The pure cultures were sub-cultured into several new plates of PDAS and maintained for use. Characters like color of culture, growth pattern, and type of fruiting structure were examined. Slides were also made from the pure cultures and examined under a stereomicroscope for characterization of the organism. These characters were used in conjunction with reference publications to identify the organism. Cultures of the organism and infected leaves were sent to Global Plant Clinic in United Kingdom for confirmation of the identity of the pathogen. This isolation was done on both red and green Roselle collected from four locations of Katsina-Ala, Gboko, Vandeikya and Makurdi Local Government Areas in Benue State, central Nigeria. The result was used to confirm whether it is the same organism that is causing this disease at these different locations and in these three accessions of the plant. Preparation of spore suspension Two weeks old pure cultures were scooped into an electric blender and 250ml of sterile distilled water was added and blended for 15 minutes to expose the spores that were held inside fruiting bodies or the pycnidia. The suspension obtained was filtered using sterile cheesecloth to remove mycelial fragments. The spore concentration was adjusted to 78 x104 spores/ml of the isolate using a haemocytometer. Inoculation of healthy plants Seeds of three accessions were planted on the 20th of July 2013 in polythene bags and allowed to grow for two weeks. They were thinned to two per bag and allowed to grow for additional two weeks. Each accession received four treatments of sprayed intact leaves with water (WIN), sprayed injured leaves with water (WIJ), sprayed intact leaves with inoculums (IIN) and sprayed injured leaves with inoculums (IIJ). This was replicated three times given a total of twelve observations arranged in a complete randomized design (CRD) in the screen house. The plants were covered for 48 hours with a black polythene sheet to increase humidity and enhance infection. The inoculated plants were observed daily for the development of the symptoms. Disease severity rating was taken after 48 hours and one week. Infected leaves were collected and the pathogens re-isolated and compared with the original isolates. The inoculum was considered pathogenic with the severity rating of between 2 and 5 as described by Mohanan et al. (1989) as shown in Table 1. In every case, a score of 5 represented total collapse of all leaves and death of the plant.

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Table 1: Table of Disease Descriptive scale. Disease Score %of leaves with symptoms Remarks 1 0 No infection 2 1-20 Slight infection 3 21-50 Moderate infection 4 51-70 Severe infection 5 71-100 Very severe infection

Data analysis All data collected were subjected to analysis of variance using GenStat 5 version 3.2, 1995 (Laws Agricultural trust: Rothamsted Experimental Station, UK)

Results and Discussion

This study identified the causal agent as being Coniella musaiensis var. hibisci which was confirmed by the Global plant clinic in the United Kingdom. Cultural characteristics were used in the identification of the causal agent. A pure culture of the fungus on PDAS (Plates 1) is whitish. The mycelia are profusely branched and septate as shown in Plate 2. Old cultures of the fungus produced spiny globolous dark pycnidia (Plate 3) which houses rod shaped spores (plate 4)

Plate 1.Pure culture of C. musaiensis

Plate 2: Coniella Mycelia (40 x) on Potato Dextrose Agar Supplemented with Streptomycin Sulphate

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Plate 3: Coniella Pycnidia (40 x)

Plate 4. Coniella Spores (40 x) The fungus had uniform radial growth pattern on PDAS with average colony diameter measuring 1.9 mm in a day and reaching 13.5 mm in 7 days. The fungal colonies causing the observed leaf spot disease of roselle in Benue State was believed to survive in the soil, seeds as well as in debris from infested fields. C. musaiensis var. hibisci appeared to be ubiquitous as was observed infecting Roselle and causing severe disease in the field. Persad and Fortune (1989) had earlier reported C. musaiensis var. hibisci as the causal agent of leaf spot and stem canker observed on Roselle in . Adeoti and Emechebe (1996) reported the same pathogen as being causal agent of leaf spot disease observed in Kenaf (Hibiscus cannabinus L.) where it was even tagged the most important disease of the plant in Nigeria.

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Pathogenicity Test Disease severity was rated after 48 hours and 1 week of inoculation and presented in Table 2.The result from this study showed significant difference among the treatments but none was shown between the accessions at both 48 hours and after one week. Disease severity in leaves (Plate 5) injured and treated with the pathogen was significantly (p=0.05) higher than in the rest of the other treatments as shown in Table 2, again for both 48 hours and after one week of treatment. Symptoms manifested within 24 hours of inoculation. Spots were also observed in both the injured and intact leaves treated with water after one week of treatment; however the organism was not re-isolated from the spots. It was also noticed that disease did not spread to newly emerged leaves after the dropping of the infected ones. This points to the fact that the pathogen on its own can’t penetrate the plant epidermal wall and relies on external factor(s) to inflict an opening for it to have access to the internal tissue.

Plate 5. Inoculated Plants showing Symptoms on Leaves

Table 2. Disease Severity Rating of three Inoculated Roselle Accessions in the Screen House.

Inoculation period 48 hours 1 week Treatment WIN 1.00 1.22 WIJ 1.00 1.56 IIN 1.00 1.89 IIJ 1.67 3.00 Accession 1 1.08 1.67 3 1.17 2.00 4 1.25 2.08 LSD (P=0.05) Treatment 0.23 ⃰ 0.49 ⃰ LSD (P=0.05) Accession 0.20 ns 0.42ns

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Where:- WIN=Intact leaves with water WIJ=Injured leaves with water IIN=Intact leaves with Pathogen IIJ=Injured leaves with pathogen LSD = least significant difference ns = indicates not significant at 5% * =Significant at 5%

Conclusion

Successful identification of disease and causal agent coupled with sources of inoculum are basic efforts towards control of such disease. This study identified the causal agent of leaf spot disease of roselle as being Coniella musaiensis var. hibisci and it was also found that the pathogen lives in the soil, seeds as well as in debris from infected fields. It also points out that the pathogen is a wound pathogen as it was unable to attack intact leaves.

References

Adeoti, A.A & Emechebe,A.M., (1996). Effect of Sowing Date and Type of Fertilizer on Kenaf Yield and its Reaction to Coniella Leaf Spot. Samaru Journal of Agricultural Research, 13: 13-18. Agrios, G.N., (2005). Plant Pathology (5th Edition).Elsevier- Academic press San Diego, C.A. 922pp. Alegbejo, M.D., (2000). The potential of roselle as an industrial crop in Nigeria. NOMA, IAR Samaru, Zaria, 14: 1-3. Collins, C.H. & Lyne, P.M., (2004). Microbiological Methods. Arnold, London 465pp. Mohanan, R.C., Kaveriappa, K.M & Nambiar, K.K.N., (1989).Epidemiological studies of Collitotricum gloeosporioides disease of cocoa. Annals of Applied Biology, 114(1):15- 22. Morton, J., (1987). Roselle p281-286. In of warm climate. Julia F. Morton, Miami,FL. http://www.hort.purdue.edu/newcrop/mortonne/roselle.html Access 11 August 2017 Onyenekwe, P.C., (1998). Antihypertensive effect of roselle Hibiscus sabdariffa calyx infusion in spontaneous hypertensive rat and comparison of its toxicity with that in water rats. In: Journal of Biochemistry and cell Function, 11:120-126. Persad, C. & Fortune, M., (1989). A new disease of (Hibiscus sabdariffa var. sabdariffa) caused by Coniella musaiensis var hibisci from Trinidad and Tobago. Plant Pathology, 38:615-617. Qi,Y., Chin, K.L., Malekian, F., Berhane, M. & Gager, J., (2005). Biological characteristics, nutritional and medicinal value of roselle Hibiscus sabdariffa L. Circular-Urban Forestry Natural Resources and Environment No.604. Schippers, R.R., (2000). African indigenous vegetables: An overview of the cultivated species NRI Greenwich 214pp. Sharoff, A., (1996). The pharmacological characteristics of H. Sabdariffa. Planta Medical (Stuttgart), 10: 48-52. Tindall, H. D., (1983). Vegetables in the tropics. Macmillan press ltd. London 734pp.

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