First report of citrus bacterial canker disease in lime (Citrus aurantifolia Swingle) in Gadaref State-Eastern Sudan

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1 AGRICULTURE AND BIOLOGY JOURNAL OF NORTH AMERICA ISSN Print: , ISSN Online: , doi: /abjna , ScienceHuβ, First report of citrus bacterial canker disease in lime (Citrus aurantifolia Swingle) in Gadaref State-Eastern Sudan M. Y. A. Abubaker 1, O. A. B. Abu Dibar 2, S. M. Elhassan 2, N. M. E. Yousif 2 1 Department of Pests and Plant Health, College of Agriculture, University of Bahri, Khartoum, Sudan 2 Department of Crop Protection, Faculty of Agriculture, University of Khartoum, Shambat, Sudan ABSTRACT Field surveys were conducted during the rainy season in commercial citrus orchards, nurseries, and home backyard citrus trees in three localities (Gadaref, Mafaza, and Fashaga) in Gadaref State to investigate the possible occurrence of citrus bacterial canker disease (CBCD). Typical symptoms of CBCD were produced naturally on lime trees only consisting of erumpent corky lesions surrounded by water-soaked tissues and a yellow halo on leaves, fruits and branches. In severe infections, fruit lesions with crater-like centres, frequent fruit drop, extensive defoliation and dieback of twigs and branches were commonly evident. Gadaref and Mafaza localities recorded a disease incidence of 65.34% and 8.68%, respectively, while no CBCD was recorded in Fashaga locality during these surveys. Home backyard trees recorded the highest incidence (45.9%) followed by commercial orchards (25.3%), whereas nurseries exhibited the least disease incidence (17.0%). Citrus canker bacterial isolates originally recovered from naturally infected lime leaves reproduced typical citrus bacterial canker (CBC) lesions when inoculated with a high inoculum concentration (10 8 CFUml -1 ) on both detached and attached leaves of selected citrus varieties. Similar lesions with varying pathogenicity scores developed on lime, grapefruit and mandarin test leaves but not on orange and other citrus varieties. Bacteria was isolated from various infected citrus plant parts, and phenotypically characterized. The survey results in addition to the pathogenicity, morphological, biochemical, and growth characteristics-tests indicate the occurrence of CBC in eastern Sudan. This represents the first report of CBCD in Sudan. Key words: Citrus; Citrus bacterial canker; Sudan. INTRODUCTION Sudan lies within the citrus belt and appears to possess enormous potentialities in Citriculture. The commercial citrus production spreads all over the country including Gadaref State in eastern Sudan (Mahdi, 1979). The total area of citrus production is estimated over 170,000 ha producing annually around 2,400,000 metric tons with a sizable export, mainly to the Middle East countries and Turkey (National Horticulture Administration, 2013). Citrus bacterial canker disease (CBCD) is one of the most feared of citrus diseases affecting almost all citrus varieties. It is wide spread in citrus producing areas of the tropical and sub-tropical world causing severe canker lesions on all above-ground parts of the plant, leaf defoliation, premature fruit drop, tree decline and reduced fruit marketability (Roistacher and Civerolo, 1989). In addition, it has a negative impact on the international trade. Consequently, it has particularly been managed through eradication procedures and strict quarantine regulations (Gottwald et al, 2002) beside other chemical and cultural practices (Krishna and Nema, 1983 and El- Goorani, 1989). The Asiatic and the atypical strains of the pathotype A (A and A * strains) are widely prevailing in South-West Asia, South-East Asia, the horn of Africa and some other African countries in West Africa (Alizadeh et al., 1990; Al-Saleh et al., 2014; Bui et al., 2008; Derso et al., 2009; El-Goorani 1989; Juhasz et al., 2013). However, CBCD has not been reported in Sudan before autumn 2013 (Elhassan et al., 2014; Eppo, 2013) although some extensive surveys have previously been conducted to establish the possible occurrence of this disease. Typical disease symptoms were observed for the first

2 time in Gadaref State during the rainy season in 2013, only on lime (Citrus aurantifolia Swingle), but no symptomatic trees were encountered in canker susceptible grapefruit and orange varieties grown in the same orchards. This paper is intended to survey the natural occurrence of citrus canker in Gadaref commercial citrus orchards, nurseries and home backyard citrus trees and to establish the identity of citrus canker bacterium pathotype strains based on phenotypic and biochemical characteristics, as well as on the pathogenicity tests of the recovered isolates. MATERIALS AND METHODS Field survey: Three field surveys were carried out towards the end of the rainy season of the years 2013 and 2014 (September-October) in some citrus groves of Gadaref state. Each survey included three citrus planting types, namely, commercial orchards, nurseries and home backyard trees. The first survey was conducted in Gadaref locality and the other two were extended to Fashaga and Mafaza localities (Fig.1). For each survey CBCD incidence (DI), and CBCD severity were evaluated (DS). Fig.1. Map of Gadaref State showing the surveyed areas [ Mafaza localities. ] of Gadaref, Fashaga and 263

3 Each locality was divided into three main areas (site1, site2, site3). At each site 3-5 commercial orchards and nurseries were randomly chosen and about trees of available citrus varieties were inspected for the presence of CBCD. A total of 200 lime trees and 30 to 100 orange and grapefruit trees were examined in each locality. All nursery seedlings and backyard citrus trees found at the site were inspected for CBCD incidence (DI). The percentage of infection was recorded for each planting type at each locality and the data were statistically analyzed. The calculations were based on the following formula: For disease severity (DS), ten infected trees were randomly selected from each planting type at each locality. Symptoms on the leaves, fruits and twigs were examined visually and described. The severity was recorded at the specified dates of the survey according to the following scale (SS): 1= leaves on few {1-2} branches showing symptoms. 2= leaves on several (up to 10%) branches showing symptoms. 3= leaves on many (>10-25%) branches showing symptoms plus symptoms on few fruits and twigs. 4= >25-50% of the tree canopy showing symptoms on leaves, fruits and twigs. 5= > 50% of the tree canopy showing clear canker symptoms on leaves, twigs, fruits, main branches and trunks. In addition, severe defoliation and die-back are evident. The disease severity (DS) was then calculated as follows: The data were then statistically analyzed using the SAS System for ANOVA table. Laboratory tests: i) Isolation and purification Isolation and purification of bacteria from infected leaves, fruits and twigs were conducted following the National Diagnostic Protocol (NDP) for asiatic citrus canker (2014). The bacteria were grown on nutrient agar (NA). ii) Phenotypic Characterization The biochemical tests; as Gram staining reaction, starch hydrolysis, oxidase test, and catalase test, were carried out according to Kidist (2003). Whereas motility, ability to grow under anaerobic conditions and growth at 36 C were carried out according to Cowan (1974) and Fahy and Persley (1983). iii) Pathogenicity tests on detached and attached leaves RESULTS Field survey Pathotypes and strains of Xanthomonas citri subsp. citri can be defined based on their pathogenicity to a range of citrus cultivars. The test was performed essentially according to the method described by the NDP protocol for asiatic citrus canker (2014). Acid lime (local variety), Eureka lemon (Kaffir lime), Cleopatra mandarin, orange (Nouri 16) and grapefruit (Red blush) used in these tests were kindly supplied by the Department of Horticulture, Faculty of Agriculture, University of Khartoum. i) Symptomatology The survey conducted in three localities in Gadaref State in commercial citrus orchards and nurseries in addition to home backyard citrus trees displayed some characteristic symptoms of CBCD on foliage, fruits, twigs and branches of local lime (Citrus aurantifolia Swingle), but not on other citrus varieties. Typical symptoms were observed upon close examination of symptomatic local lime trees. The characteristic external symptoms were necrotic lesions consisting of erumpent corky tissues on leaves, fruits, branches, twigs and thorns (Fig. 2 and Fig.3). The canker lesions on leaves and fruits were often surrounded by water-soaked tissues. Raised corky or erumpent lesions appeared on leaves surrounded with a prominent yellow halo. 264

4 A B C D Fig.2. Infected lime Leaves showing typical symptoms of citrus bacterial canker disease. A. Erumpent lesions with yellow halo. B. Raised corky young lesions surrounded by yellow halo on upper leaf surface. C. Corky lesions on lower leaf surface. D. Unusual citrus canker lesions associated with leafminer damage Unusual canker lesions developed on the leaves. These were associated with mechanical (i.e. thorn) and leaf miner damages in which many canker lesions coalesced to form elongate or blotchy corky patterns on affected leaves (Fig. 2). Canker lesions with erumpent corky tissues were also found sparsely distributed or largely covering small twigs and branches of affected lime trees. In severe cases of foliage infection extensive leaf defoliation and dieback symptoms were commonly observed. The fruits were particularly susceptible to the canker pathogen, often developing severe canker lesions with craterlike centers. Such infections ultimately lead to piles of fallen fruits underneath the affected lime tree (Fig. 3). ii) Disease Incidence (DI) and Disease severity (DS) of CBC: The results are shown in Tables (1 and 2) and Figures (2 and 3). Strikingly high CBCD incidence levels were recorded in Gadaref locality in all of the three planting types (100%, 88.7% and 38.3% for backyard trees, orchards and nurseries, respectively), with an overall locality mean of 75.7%. In Mafaza locality, CBCD was only present in commercial orchards (9.7%), while it was not detected in any of the three planting types in Fashaga locality. Significant differences in disease levels were evident between localities as well as between planting types, where CBCD incidence was significantly higher in backyard (30.6%) and commercial orchards (30.09%) trees over that in nursery stocks (13.95%). Same trends of CBCD severity levels were observed with respect to variations among localities and planting types. Home backyard trees in Gadaref locality recorded the highest severity (2.4), followed by commercial orchards in the same locality (2.2) while the nursery stocks, which were only found infected in Gadaref locality showed the least severity 265

5 A B C D Fig. 3. Symptoms of citrus canker on different parts of lime tree: A: Canker lesions on lime fruits. B: a pile of fallen fruits affected by citrus canker. C: Canker lesions on twigs and branches. D: Leaf defoliation and die-back symptoms of citrus canker infection. 266

6 A Fig. 4. Cultural and morphological characteristics of Xcc-Gadaref isolate A: Pale yellow colonies on nutrient agar medium B: Rod-shaped citrus canker bacteria seen under the light microscope (100X). B (1.4). The disease was much more severe in Gadaref locality as compared to that in Mafaza. Characterization of citrus canker bacterium: A Presumptive Xanthomonas citri subsp citri- was isolated from different citrus plant parts. i) Phenotypic characteristics a. Morphological Characteristics: Colony growth of Gadaref isolate of citrus canker bacterium appeared pale yellow and very mucoid on NA and had a "sticky" texture when touched with a loop (Fig. 4). Smears of cultures stained for Gram reaction revealed that these isolates were Gram-negative, short rods and motile (Fig 4). b. Biochemical and physiological Characteristics: Table 3 summarizes some key tests of the biochemical and physiological characteristics of citrus canker bacterium-gadaref isolate. They had negative oxidase reaction, positive catalase and they were aerobic and had the capability to hydrolyze starch and grow at 36 C. iii) Pathogenicity tests: The detached and the attached leaves of the test citrus varieties reacted compatibly at high inoculums concentrations (10 8 CFU/ml) but with different degrees of susceptibility to infection with the test bacteria of Gadaref state isolate, especially during the early days of incubation. 267

7 Local lime, Cleopatra mandarin and Red blush grapefruit seemed to be the most susceptible with varying pathogenicity scores during the incubation periods (Figs 5 and 6), while very little or no lesions were illustrated on the test citrus varieties such as Eureka lemon and orange. Fig.5. Reaction on local Lime leaves inoculated with Gadaref isolate of citrus canker bacterium. Upper: Beginning of callus formation, and/or crystalline callus at sites of inoculation after five days; pathogenicity score 2. Lower: Advanced tan colored callus with chlorosis and water soaking after 23 days; pathogenicity score

8 Fig. 6. Reaction on Cleopatra mandarin and grapefruit inoculated with Xcc-Gadaref isolate. Upper: Reaction at day 23 on Cleopatra mandarin. Advanced tan-coloured callus with chlorosis and water soaking; pathogenicity score 4. Lower: Reaction at day 8 on Grape fruit Red blush. White crystalline callus developed on all inoculation points; pathogenicity score

9 Table 1. Incidence of citrus bacterial canker (CBC) in three localities in Gadaref State (Sept- Oct 2013). Locality Disease incidence (%) on different planting types Orchard Nursery Backyard S1 S2 S3 Mean S1 S2 S3 Mean S1 S2 S3 Mean Locality mean (%) Gadarif 59.54(74.3) 82.29(98.2) 75.23(93.5) 70.36(88.7) 56.79(70.0) 42.42(45.5) 1.81(0) 38.23(38.3) 90.0(100) 90.0(100) 90.0(100) 90.0(100) 60.47(75.7) Fashaga 1.81(0) 1.81(0) 1.81(0) 1.81(0) 1.81(0) 1.81(0) 1.81(0) 1.81(0) 1.81(0) 1.81(0) 1.81(0) 1.81(0) 1.81(0) Mafaza 1.81(0) 24.12(16.7) 20.70(12.5) 18.1(9.7) 1.81(0) 1.81(0) 1.81(0) 1.81(0) (4.9) Figures between parentheses are actual incidences. Actual percent values were transformed to Arc sin. Means within a column followed by the same letters are not statistically different at P : No such planting type exists. 270

10 Table 2. Severity of citrus canker disease in different localities in Gadaref State (Sept-Oct 2013 Locality planting types Orchard Nursery Backyard Locality mean Gadaref 2.17a 1.40a 2.37a 1.98A Fashaga 0.0b 0.0b 0.0b 0.0B Mafaza 0.1b 0.0b B Mean of planting type 0.76AB 0.47B 1.18A Severity scale of 1 to 5 was used. Means within a column followed by the same letters are not statistically different at P : No such planting type exists, in Mafaza locality. 271

11 Table3. Biochemical and physiological characteristics of Xanthomonas citri subsp citri- Gadaref isolate Characteristic noitcaer Gram reaction Motility KOH solubility Catalase Cytochrome C oxidase Growth at 36 Cº Aerobic test Hydrolysis of starch Gelatin hydrolysis Casein Tween 20, Tween 80 YDC - - Mucoid 272

12 DISCUSSION The present results reveal the occurrence of a newly emerging citrus bacterial canker disease (CBCD) naturally spreading in lime trees (C. aurantifolia Swingle) in Gadaref State in eastern Sudan. It appears to be a distinctive strain of citrus canker bacteria. The fact that other citrus varieties (especially grapefruit and sweet orange) in the surveyed areas, even in close proximity to lime, did not show any citrus canker symptoms was an indication of the host specificity of the causal agent. The host range restriction of CBCD to lime has also previously been reported from Maldives islands (Roistacher and Civerolo, 1989), South-West Asia (Verniere et al., 1998), Thailand and Cambodia (Bui et al., 2007; 2008), Ethiopia (Derso et al., 2009) and recently again from the western region of Saudi Arabia (Al-Saleh et al., 2014). These were collectively designated as Xcc-A *. Although closely related to Xcc-A (the Asiatic type of canker), these strains can be readily distinguished from the known strains of Xcc-A based on their atypical combination of host range and symptomatology (Verniere et al., 1998). The present Gadaref isolate appears to resemble closely these strains of the pathotype Xcc-A *, at least in the phenotypic characterization and some cultural and biochemical characteristics. However, the reason for the susceptibility of detached and attached leaves of grapefruit and citrus varieties other than lime to citrus canker bacterium-gadaref isolate is intriguing, but could be due to the delay in suberization of host cell walls, decline in the host defense reactions (Mohammadi et al., 2001) or to the high inoculums concentrations used in the test. Another unique strain of citrus canker bacteria from Mexican lime and Alemow in South Florida designated Xcc- A w (Sun, et al., 2004) appears to be more related to Xcc-A* strains and thus also furnishes some similarities to Gadaref isolate. It could be inferred that these strains of citrus canker bacteria restricted to lime only or mainly such as Xcc- A*, Xcc- A w, Xcc-Maldives strains and Xcc- Gadaref isolate are members of a larger group of strains originated in one or a close geographical territory, probably in southeast Asia (Verniere et al., 1998). This speculation could be validated by collecting and fully characterizing a large group of strains specific to lime in South West Asia, South East Asia, South Florida and Eastern Sudan. The geographical proximity and the humid southeastern wind-blown to eastern and southeastern Sudan from these areas where the atypical Asiatic (Xcc- A*) form is prevalent make Gadaref and the neighboring citrus production areas especially vulnerable to attack by variants of this pathotype. Generally, the climatic conditions of precipitation and maximum and minimum temperatures (i.e mm, o C, respectively) prevailing during July- September in Gadaref are favourable for infection, but the wind speed (1.7 m/sec) may not promote the pathogen spread to a greater extent (Roistacher and Civerolo, 1989). Collectively, the association and isolation of Xanthomonas citri from symptomatic trees, the host range, pathogenicity tests, symptomatology and the biochemical tests strongly indicate that the currently investigated disease on lime in Gadaref State (Eastern Sudan) is CBC and these lime isolates are closely related to strains of the atypical Asiatic (Xcc- A*) pathotype. Detection of considerately high level of CBCD infection in Gadaref area suggests that there is a potential large scale dissemination of the citrus canker bacteria - Gadaref isolate within the commercial citrus orchards in Gadaref State and probably to other neighboring states in eastern and southeastern Sudan. Since the wind speed is somewhat sub-optimum in this area, citrus nurseries may constitute the primary method of long distance spread of citrus canker bacteria in Gadaref and other neighboring citrus production areas (Gottwald et al., 2002 and Gottwald and Irey, 2007). However, in many other areas around the world Xcc strains spread primarily by wind-blown rain (Roistacher and Civerolo, 1989). Last fall (September- October/2014) the disease was found spreading in all Gadaref localities and in the main citrus producing areas of Kassala State (see map) (unpublished data). It appears that, the meteorological data during the fall season may be close to optimum for local spread of citrus canker bacteria in eastern Sudan. However, as contaminated citrus nursery stocks may constitute the major source of primary pathogen inoculums in eastern Sudan, sanitation of citrus nurseries against CBCD seems mandatory for effective disease management. Also, planting wind breaks around unaffected orchards may also serve as a safeguard against wind-driven rain carrying primary citrus canker bacteria inoculums when epidemiological conditions are favourable (Gottwald and Timmer, 1995). Further phenotypic and genotypic characterizations of Xcc-Gadaref isolate and other strains from Sudan are deemed essential and constitute a pre-requisite to further epidemiological studies and to regulatory programs. 273

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