Occurrence, Etiological and Ecological Aspects of Bacterial Blight of Guar in Sudan

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1 American-Eurasian J. Agric. & Environ. Sci., 18 (6): ISSN IDOSI Publications, 2018 DOI: /idosi.aejaes Occurrence, Etiological and Ecological Aspects of Bacterial Blight of Guar in Sudan Isameldin O.A. Yousif, Azza S. Abbo, Mohammed A.A. Hamad, 4 2 Muneer E.S. Eltahir and Siddig M. Elhassan 1 Department of Plant Protection, University of Kordofan. El Obeid, Sudan. 2 Department of Crop Protection, University of Khartoum, Khartoum, Sudan 3 Department of Rural Extension and Training, University of Kordofan. El Obeid, Sudan 4 Department of Extension, Training and Documentation, Institute of Gum Arabic Research and Desertification Studies, University of Kordofan. El Obeid, Sudan Abstract: The present study was conducted to investigate outbreak of a devastating wilt disease of guar (Cyamopsis tetragonoloba L.) in its commercial production areas in Blue Nile and Gadaref states in the year Two field surveys were carried out in irrigated and rain-fed production areas in years 2013 and The results indicated that the disease incidence was greatest under rain-fed conditions, where 100% infection was recorded in the Blue Nile State, followed by 74% disease incidence at Gennib location. However, in the irrigated areas the disease was very meager (0.02 and 0.5%). The affected guar plants developed leaf spots, stem lesions, stem canker and under severe infection wilting and defoliation were also evident. Three bacterial isolates were recovered according to type of symptoms and locality. The three bacterial isolates were negative to Gram staining test, motile, had the ability to grow in NA and YDC agar culture media, even at 36 C and tolerant to 3% salt and have the capability to grow aerobically, hydrolyze starch and gelatin and they were oxidase positive. The pathogenicity test availed that the original symptoms were reproduced on healthy guar seedlings verifying that the detected and identified bacterium was Xanthomonas axonopodis pv. cyamopsidis. Key words: Blight Etiology Incidence Irrigated Rain-fed Sudan INTRODUCTION grown guar for the first time in 2013, [10]. The causal bacterium of the disease was identified in Arizona as Guar (Cyamopsis tetragonoloba L.) is originated Xanthomonas campestiris pv. cyamopsidis, [11]. and developed in the arid areas of the Indo-Pakistan. Recently, Jain and Agrawal [12] and Ren et al. [10] used It needs a hot climate, has a high tolerance to salinity the name Xanthomonas axonopodis pv. cyamopsidis and a good capability to fix atmospheric nitrogen, [1-6]. (Xac). Guar plants are more susceptible to Xac at an early Guar seeds contain about 4% edible oil and a protein stage of growth (20-30 days), leading to their drying and content ranging between 27 and 37%. Recently, guar death, [13]. Leaves, stems and Guar bean pods are gum has also been studied as a substitute for fat in damaged causing substantial losses. A decrease of human food, [7]. Guar is a relatively new crop in to 25.15% in the number of pods per plant has Sudan, it has been grown commercially since 1980s in been reported as well as a reduction in the seed quality, Blue Nile, Gadaref, Kordofan and some other scattered [12, 14, 15]. areas in other states, [8]. However, still little research has This research aimed to survey the commercial fields been conducted in Sudan to study the agronomic in the main Guar production areas to assess the incidence response of guar and its potential as cash and industrial of bacterial blight disease, to study the disease symptoms crop, as well as its important diseases and their developing during the crop lifecycle under different management. Bacterial blight of guar has been reported cropping systems and to isolates, characterize and test firstly from the State of Rajasthan in India as early the pathogenicity of the Sudanese isolates of the causal as1950s, [9]. In China, the disease was observed in field- bacterium. Corresponding Author: Azza S. Abbo, Department of Crop Protection, Faculty of Agriculture, University of Khartoum, Khartoum, Sudan. 358

2 MATERIALS AND METHODS Total of three bacterial isolates were obtained from yellow bacterial colonies recovered from stem lesions Disease Survey: Surveys were carried out in the main at Aelaphon (ALY) and stem lesions & canker, production areas of guar in Sudan during two growing respectively, from Genneb (GLY and GCY). Unfortunately, seasons at pod stage. The first survey was conducting in isolation from samples collected from Grabeen and Sennar 2013 in Blue Nile State (four fields at Grabeen), while the areas were ignored in this study because they were second survey was conducted in 2014 in Sinnar State completely dead and highly contaminated. Stocks of pure (Debaleeba and Abosibeaha), Gezira State (Genneb) and cultures of the bacterial isolates were maintained on NA Khartoum State (Aelaphon). Three sites per field were medium. randomly taken and 100 plants per site were inspected for symptoms development. Morphological and Cultural Characteristics: The isolated bacteria were inoculated on NA media plates and Isolation of the Causal Pathogen: The pathogen was incubated at 26 C. Cultural characteristics such as type of isolated from stems of symptomatic plants showing either colony growth and colour were examined and described. stem lesions or stem canker. Tissues of stem lesions A thinly film of bacterial suspension was made on a clean showing canker were cut into small bits, thoroughly slide for Gram staining test. The motility of the bacterial washed with tap water, surface-sterilized with 6% Sodium cells was examined by placing a drop of sterile water in the hypochlorite, rinsed in three changes of sterilized distilled middle of a clean cover slip. A loopful of the bacterial water and excess water was removed by placing stem growth was suspended in the drop of water. A tiny drop tissues in sterile filter paper. Stem bits were then of mineral oil was placed on each of the four corners of suspended in 10 ml sterilized distilled water for two hours the cover slip, then the cover slip was reversed and before streaking was performed onto nutrient agar (NA) placed over the slide. The slide was observed under the using sterilized loops. light microscope with the oil immersion lens (x100 Subcultures on NA were made from discrete colonies magnification), [16]. of the dominant bacterial growth to obtain pure cultures of the pathogen. Establishment of pure line culture of the Biochemical Characteristics: The biochemical bacterial isolates was attempted by 3-5 serial inoculation characterization of the bacterial isolates was carried out of a typical discrete lesion onto NA plates. using essentially the methods described by Hayward [17]. Table 1: Incidence and symptoms of bacterial blight of Guar surveyed in some rain-fed and irrigated commercial farms State and location Field area (feds) Season Type of irrigation Seed source Symptoms Disease incidence Blue Nile Grabeen field Autumn 2013 Rain-fed Imported commercial Leaf spots Stem canker Grabeen field Autumn 2013 Rain-fed Imported commercial Leaf spots Stem canker Grabeen field Autumn 2013 Rain-fed Imported commercial Leaf spots Stem canker Grabeen field Autumn 2013 Rain-fed Imported commercial Leaf spots Stem canker Sinnar Dibaleeba 05 Autumn 2014 Rain-fed+Furrow Faculty of Agriculture Leaf spots wilt 0.3% irrigation Abosibeaha 03 Autumn 2014 Rain-fed Imported commercial 0.00% seed stock Gezeira Genneb field 1 03 Autumn 2014 Furrow irrigation Faculty of Agriculture Leaf spots Stem canker 100% wilt Genneb field 2 18 Autumn 2014 Furrow irrigation Faculty of Agriculture Leaf spots Stem lesion 48% Stem canker wilt Khartoum Aelaphone 03 Autumn 2014 Furrow irrigation Faculty of Agriculture Leaf spots Stem lesion 0.5% 359

3 Table 2: Biochemical characteristics of Sudanese isolates of bacterial blight of Guar Biochemical tests ELY GLY GCY Growth on NA +ve +ve +ve Gelatin hydrolysis +ve +ve +ve Growth on Y D C +ve +ve +ve Gram staining -ve -ve -ve Motility +ve +ve +ve Aerobic growth +ve +ve +ve Starch hydrolysis +ve +ve +ve Oxidase +ve +ve +ve Catalase +ve +ve +ve Growth at 37 C +ve +ve +ve Salt tolerance +ve +ve +ve Glucose utilization +ve +ve +ve Manose utilization +ve +ve +ve *ALY: Stem lesions at Aelaphon, GLY: Stem lesions at Genneb, GCY: Canker at Genneb Yeast extract dextrose calcium carbonate (YDC) plates were streaked with a loopful of the bacterium and incubated for 48 hrs at 28 C. Catalase activity of the isolates was tested by mixing a loopful of bacterial cells from young culture with a drop of hydrogen peroxide 5% (H202) solution on a slide. The Oxidase activity of the isolated bacteria was tested by mixing a loopful of the bacteria with the oxidase reagent on a filter paper. Testing the oxidation of carbon was performed by inoculating test tubes containing medium supplemented with tow carbohydrate compounds glucose and manose, two tubes were left uninoculated as control. Tubes were incubated at 28 C for 24 h. The ability of the bacteria to hydrolyze starch was examined by growing the isolated bacteria on nutrient agar starch (NAS) medium. The bacterial isolates were tested for anaerobic growth by inoculating these bacteria into test tubes containing NA medium before solidification of the medium, tubes were then incubated at 28 C for 24 h. Test tubes containing commercial nutrient gelatin (NG) were used to test the presence or absence of proteolytic enzymes in the isolated bacteria. The tubes were inoculated by the bacterial isolates two tubes were left uninoculated as control. Tubes were incubated at 28 C for 24 h then cooled to 3 C for 15 min and incubated for 7 days at 28 C. Salt tolerance of these bacteria was examined by inoculating the isolates in nutrient broth cultures supplemented with respectively, 2, 2.5 and 3% NaCl. Turbidity of cultures was checked 24 h after incubation at 28 C. Heat tolerance of these bacteria was examined by streaking the isolates on NA plates and incubated them at 36 C for three days. Pathogenicity Test: In vivo pathogenicity test of the isolated bacteria was done in April 2015 following Ren et al. [10]. By: (i) spraying the bacterial suspension on the leaves until runoff and by (ii) injection into the stem using 5-cc- sterile syringe and equal number of plants were treated with sterile distilled water as control. Plants were kept moist by placing under a plastic sheet cover in a shaded nursery. The humidity was enhanced by applying frequent mist during the day in addition to flooding of the ground underneath the pots. Symptoms development was then observed. Re-isolation was performed from the developing lesions and cankers in NA medium to satisfy Koch s postulates. RESULTS Field Survey for Disease Incidence and Symptoms: Table 1 summarizes the results of the survey conducted at different locations for bacterial blight of Guar. It includes field area, season, type of irrigation, seed source and symptoms & disease incidence. The disease incidence was 100% at Grabeen area in Blue Nile State in season It was remarkably high at Genneb in Geziera State in season 2014, where it ranged between % with a mean disease incidence of 74%. Negligible disease incidence ( %) was observed in other areas in Sinnar and Khartoum states. Leaf spots were the most common symptoms observed at all locations, which appeared on affected plants at the pod stage. All the bacterial blight syndrome; leaf spots, stem lesions, stem canker, defoliation and wilt were observed at Grabeen in the Blue Nile State and to a lesser extend at Genneb in Gezeira State. However, leaf spots and wilt symptoms, but no stem canker, were observed in Sennar, while at Aelaphon milder symptoms consisting of leaf spots and stem lesions were developed. Identification of the Causal Pathogen: The bacteria were constantly isolated from stem lesions and cankers of symptomatic guar plants in NA medium. Three isolates of the bacteria were obtained, according to selected location and type of symptoms. These were Aelaphon isolate (ALY), Genneb isolate (GLY) and Grabeen isolate (GCY). The bacterial isolates produced mucoid yellow colonies on NA and YDC media, they showed Gram-negative characteristic. They were motile. Catalase 360

4 activity of the isolates was positively detected as identity of the isolated bacterium as Xanthomonas indicated by the gas being evolved. The isolates changed axonopodis pv. cyamopsidis The pathogenicity test and the color from green to yellow indicating positive for the typical bacterial blight symptoms further oxidation test and from blue to yellow indicated their substantiated the etiology of the pathogen. ability to hydrolyze glucose and manose. The isolated The markedly high disease incidences recorded in bacteria showed clear zones of hydrolysis, which the Blue Nile State (100%) and in the northern Gezeira indicated positive reaction for starch hydrolysis. State at Genneb (74%) are expected based largely on the The inoculated bacteria in NA tubes before solidification conducive epidemiological conditions. Being a bacterial of the medium failed to grow anaerobically. After 7 days disease, the excessive wind-driven rains and the high of incubation, the isolated bacteria did not liquefy gelatin relative humidity constantly encountered during the first media when compared with the control; hence they were half of the rainy season while guar plants are still young negative for gelatin hydrolysis. All tested isolates were have evidently caused the complete infection of the crop found to tolerate the high salt concentration up to 3% in the Blue Nile State. The disease incidence at Genneb NaCl and the high temperatures up to 36 C. These results although attained a considerably high level, but at one indicated that, all the isolated bacteria from the diseased location it was not as severe as that in the Blue Nile State. guar plants under study were Xanthomonas axonopodis No defoliation or death of plants had occurred and this pv. cyamopsidis. could be attributed to the fact that it was sown in August when most of the conducive conditions had already Pathogenicity Test: Stem and leaf symptoms similar to passed in July. However, the other location at Genneb those of the original plant symptoms were reproduced on recorded 100% infection which was also more severe the inoculated guar plants within 10 days of inoculation. since it was sown early in July when the conditions were Leaf spots were the first symptoms to appear as more appropriate for bacterial blight spread and water-soaked spots, brown in colour, then the colour development. The very meager disease incidence became dark and the infected leaves fell off. Symptoms recorded at Aelaphon' and Sennar could be ascribed on the stem started as lesions and then developed to stem mainly to the relatively dry condition in the former areas canker. No symptoms developed on sterile water injected and to late sowing (September) of the crop in the latter plants. Yellow bacterial colonies were re-isolated from one. These conditions are suppressive for disease symptomatic plant tissues and they demonstrated development. These findings agree with Mihail and identical culture characteristics to the original culture of Alcorn [11] who reported the occurrence of a high the isolates under test. incidence of bacterial blight of guar in Arizona State after rainfall. Recently, Ren et al. [10] reported the first DISCUSSION occurrence of bacterial blight of guar in China and noticed the dependence of disease development on humidity and A devastating epidemic disease of guar was frequent rains. encountered during the rainy season of the year 2013 in In addition, contrary to the conducive ecological vast commercial production areas of the Blue Nile and factors prevailing in the Blue Nile State in the terms of Gadaref states. It has a serious impact on growth of guar continuous guar cultivation in the same area, presence of plants causing wilt and excessive defoliation and alternative weed reservoirs (such as?senna') and consequently an almost complete crop yield failure. contaminated seed source commonly recycled in this The present study revealed that this naturally spreading traditional guar production area, the other two areas at disease is the bacterial blight of guar as indicated by the Genneb and Aelaphon used clean seed source in field visual symptoms of the symptomatic plants, soils which had never before witnessed guar cultivation. characterization of the isolated bacterium and The ecological significance of the seed-born inoculum pathogenicity tests on one month-old guar seedlings. of bacterial blight of guar was reported by Jain and The typical cultural growth characteristics of the various Agrawal [12]. They found that it plays a major role in the isolates recovered from stem lesions and stem cankers of disease transmission from seed to the growing crop. It affected guar plants coupled with the biochemical tests may further build up the bacterial inoculum in the fields agreed precisely with published description of Xac by and aggravates the epidemic situation as has happened in Ren et al. [10] and Srinivasa et al. [18] and established the the Blue Nile disease outbreak model. 361

5 The wide array of symptoms development comprising 7. Zambrano, F., P. Despinoy, R.C.S.C. Ormenese leaf spots, stem lesions, stem canker, wilt, defoliation and and E.V. Faria, The use of guar and xanthan sometimes death of the effected plants were clearly gums in the production of light low fat cakes. noticed only under the conducive conditions of the Blue International Journal of Food Science and Nile production area. The nonconductive conditions of Technology, 39: Khartoum could not yield more than leaf spotting and 8. Osman, M.E., Performance of Guar as a New stem lesions. However, this has the connotation that the Crop under Flood Irrigationin Gash Dulta. In: bacterial blight of guar may not constitute a serious threat th th Proceedings of the 37 and 38 Meetings of the or constraint to expansion of the crop in nontraditional National Crop Husbandry Committee, pp: irrigated dry areas in Sudan. 9. Patel, M.K., G.W. Dhande and Y.S. Kulkarni, Bacterial leaf-spot of (Cyamopsis tetragonoloba L.) CONCLUSION Taub. Current Science, 22: Ren, Y.Z., Y.L. Yue, G.X. Jin and Q. Du, First Few perspectives seem to emerge from the present Report of Bacterial Blight of Guar Caused by research. Firstly, occurrence of the bacterial blight Xanthomonas axonopodis pv. cyamopsidis in China. consisting mainly of leaf spots, stem lesion and stem Phytopathology, 98(6): canker, leading in severe cases to wilting, defoliation and 11. Mihail, J.D. and S.M. Alcorn, Bacterial blight ultimate plant death is evident in the main growing areas (Xanthomonas campestris pv. cyamopsidis) of guar of guar in Sudan. Secondly, while devastating outbreaks in Arizona. Plant Disease, 69: 811. of bacterial blight may not be uncommon under rain-fed 12. Jain, R and K. Agrawal, Incidence and seed cultivation in the traditional production areas of guar, this transmission of Xanthomonas axonopodis pv. disease is anticipated to have a minor economic cyamopsidis in cluster bean. Journal of Agricultural importance, if any, under different cropping systems Technology, 7(1): elsewhere in Sudan. 13. Singh, S.K. and A. Mustard, An analysis of performance of Guar crop in India, National REFERENCES Institute of Agricultural Marketing (NIMA), New Delhi. 1. Whistler, R.L. and T. Hymowitz, Guar: 14. Gandhi, S.K. and J.N. Chand, Yield losses in agronomy, production, industrial use and nutrition. guar due to bacterial blight caused by Xanthomonas Purdue University Press, West Lafayette, IN, pp: 130. campestris pv. cyamopsidis. Indian Phytopathology, 2. Alexander, W.L., D.A. Bucks and R.A. Backhaus, 38: Irrigation water management for guar seed 15. Grifee, P., Crop guar plant. Food and production. Agronomy Journal, 80: Agriculture Organization of The United Nations 3. Francois, L.E., T.J. Donovan and E.V. Maas, (FAO). Salinity effects on emergence, vegetative growth and 16. Buchanan, R.E., Bergey's Manual of Systemic seed yield of guar. Agronomy Journal, 82: Bacteriology. Volumes I-IV. Wilkins and Wilkins, 4. Omer E.A., A. Fattah, M. Razin and S.S. Ahmed, Baltimore, pp: Effect of cutting, phosphorus and potassium 17. Hayward, A.C., Characteristics of Pseudomonas fertilization on guar (Cyamopsis tetragonoloba) in solanacearum. Journal of Applied Bacteriology, 27: newly reclaimed soil in Egyptian. Plant Food for Human Nutrition, 44: Srinivasa, C., U. Sharanaiah and C. Shivamallu, Elsheikh, E.A.E. and K.A. Ibrahim, The effect of Molecular detection of plant pathogenic bacteria Bradyrhizobium inoculation on yield and seed using polymerase chain reaction single-strand quality of guar (Cyamopsis tetragonoloba L.). Food conformation polymorphism. Acta Biochim Biophys Chemistiry, 65: Sin, 44(3): Ashraf, M.Y., K. Akhtar, G. Sarwar and M. Ashraf, Role of the rooting system in salt tolerance potential of different guar accessions. Agronomy for Sustainable Development, 25:

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