Characterization of fungal pathogens associated With leaf and fruit spot disease of citrus in Ethiopia

Citrus is economically important fruit crop grown throughout Ethiopia at small and commercial scales. However, citrus production and productivity in many parts of Ethiopia is seriously threatened by leaf and fruit spot disease. This disease has been reported to cause high yield and quality losses on citrus. Nevertheless, limited information have been available on the etiology of the causal pathogen and epidemiology of the disease. Therefore, surveys and laboratory works were conducted to assess the distribution, incidence and severity of leaf and fruit spot disease of citrus, and to characterize the causal pathogen under laboratory conditions using cultural, morphological, pathogenicity and molecular features. Surveys were conducted on forty-nine citrus orchards in twenty-eight districts in the major citrus growing areas of Ethiopia. Disease prevalence, incidence and severity on citrus leaves and intact fruits were determined using random sampling techniques. During the surveys, the status of citrus production and management practices were also assessed using questionnaire and field observations. Infected citrus leaves and fruits samples collected from various orchards were surface sterilized and isolated on water agar and potato dextrose agar media. Pure cultures were prepared using single spore or hyphal tip for each fungal isolate. The daily colony growth was measured and the growth rate at daily basis was calculated, while colony color and density were assessed by visual observation. Each fungal isolate culture was also evaluated for conidial and mycelial morphology by the help of stereomicroscope. Pathogenicity test for each isolate was conducted using citrus detached leaves following the standard procedure. The identity and phylogenetic relationships of the fungal isolates were analyzed using three sets of universal primers that span internal transcribed spacers, portion of long subunit region of the nuclear ribosomal DNA and partial actin gene sequences. The distribution and frequency of microsatellite loci in C. gloeosporioides genome were analyzed by generating pair-end reads from C. gloeosporioides isolate using the high-throughput Illumina sequencing platform, and reads were de novo assembled into a draft genome. Simple sequence repeat markers were developed from the draft assembled whole genome sequences of C. gloeosporioides. Fifty simple sequence repeat markers across the genome were screened using thirteen geographically representative C. gloeosporioides isolates. The polymorphic simple sequence repeat markers were used to analyze the genetic diversity and population structure of C. gloeosporioides isolates from different geographical regions of Ethiopia. The survey results showed that leaf and fruit spot disease of citrus has widely distributed in the wet humid areas of the south, southwest, central and northwest parts of Ethiopia. However, the disease was not recorded in the low moisture areas of the southeast, the central rift valley and the eastern parts of the country. The disease prevalence in the districts surveyed ranged from zero to 100%. The damage of leaf and fruit spot disease varied with citrus species and locations. The overall mean incidences and severities of the disease were highest on sweet oranges followed by mandarins. Lemons and limes were the least affected citrus species. Disease incidences and severities were higher in Jimma town, Abeshege, Aleta Wendo, Kebena, Mana, Gomma, Ginbo and Debre Werk districts. Disease incidences in the different orchards ranged from zero to 81.7% on leaves and from zero to 100% on fruits. Disease severity also varied from zero to 75% on leaves and from zero to 100% on fruits. The questionnaire assessment and field observations indicated that various citrus species (sweet orange being the major species) and varieties with two to seventy years of age were produced by smallholder and commercial farmers. The assessment also showed that diseases and insect pests, poor agronomic and irrigation practices, and shortage of adapted high yielding varieties were the major citrus production constraints in the country. In the present surveys, it has been observed that commercial citrus orchards practice field sanitation, pruning, irrigation, and fertilizer and pesticide applications. However, most of the orchards of the smallholders were not well managed. The colony characters such as color, density and daily growth rate varied among fungal isolates. The colonies varied from white to dark gray in color. The majority of the isolates produced circular, wooly or cottony colonies with pale brown or grayish white color. The fungal isolates produced colonies with compact, medium or sparse density. The average daily colony growth rate ranged from 0.04 to 2.3 cm. Some isolates were very slow-growing, whereas most cultures had characteristic fast-growing compact aerial mycelia. Majority of the fungal isolates did sporulate, but the type of conidiathey produced were not similar. These isolates produces hyaline, ovoid to oblong, slightly curved or dumbbell shaped conidia. Pathogenicity tests on detached leaf assays also revealed the association of most of the fungal isolates with foliar disease symptoms of the test citrus cultivars. Based on the multilocus analyses, more than 85% of the fungal isolates were belonged to Colletotrichum species complex (81% were C. gloeosporioides). The phylogenetic analysis of the isolates based on multilocus sequences delineated them as C. gloeosporioides sensu lato (broad sense) and C. boninense spp. complexes. Each single locus sequence analysis also identified 163 isolates as C. gloeosporioides or its teleomorph Glomerella cingulata. These findings provide information on the causal pathogen of leaf and fruit spot disease of citrus in Ethiopia and suggest the need for in-depth studies to determine the role of C. gloeosporioides species complex in leaf and fruit spot disease epidemiology. The results also demonstrated that multilocus sequences are reliable methods for phylogenetic analysis of species within the genus Colletotrichum. These findings provide baseline information for further population genetic studies of the pathogen. The information will also be useful in developing effective disease management practices against C. gloeosporioides. A genomewide microsatellite database of 5030 microsatellite motifs were identified in C. gloeosporioides genome. Of these, 94.6% were perfect motifs. Trinucleotide repeats were the most frequent; whereas penta- and hexanucleotide motifs were the least abundant. The number of motifs decreased as the number of the repeats increased. A/T repeats were more abundant than G/C repeats in the C. gloeosporioides genome. In penta- and hexanucleotide repeats, GC-rich motifs were predominant. Twenty-one simple sequence repeat markers showed polymorphism and demonstrated allele diversity among the thirteen test isolates of C. gloeosporioides. This small-scale population study could serve as a proof-of-concept showing that the genome sequencing approach was successfully applied for microsatellite discovery and development of simple sequence repeat markers. Twenty-three polymorphic simple sequence repeat markers produced a total of 118 alleles among the 163 C. gloeosporioides isolates. The polymorphic information content values ranged from slightly to highly informative. The gene diversity among the loci ranged from 0.106 to 0.664. Analysis of molecular variance showed that 85% of the total variation was due to the differences of isolates within a population. The genetic differentiation in the total populations was low as evidenced by high level of gene flow estimate (Nm=4.8) between populations. Populations of Ethiopian C. gloeosporioides from citrus were generally characterized by a low level of genetic diversity. Unweighted Neighbor-joining and population structure analyses grouped the isolates into three major clusters regardless of their geographic origins. The microsatellite markers developed and used in this study were useful to comprehend the genetic diversity and population structure of C. gloeosporioides isolates from main citrus growing regions of Ethiopia. Despite regional differences, the observed genetic diversity in all four populations was lower than expected suggesting inter-regional exchanges of planting materials and/or fruits, and dispersal of inoculum among the regions. Information generated in this study were useful in understanding the pathogen biology and provided basis for other studies on disease development, host-pathogen interaction, and developing disease management strategies for the control of leaf and fruit spot disease of citrus in Ethiopia. The SSR markers developed in this study could be used to characterize C. gloeosporioides isolates that infect other fruit crops. In conclusion, the disease should be managed at any cost. Frequent disease monitoring and precautions are essential. Care should be taken during transporting the planting materials and fruits from affected areas to locations where the disease is not recorded. Citrus growers should apply soil fertility management practices in their orchards, and practice general hygiene and sanitary measures. Application of relatively safe fungicides could reduce the disease damage. It is also necessary to investigate the reactions of the available citrus cultivars to the pathogen and select disease tolerant/resistant scions. In the long term, integrated disease management approaches including biocontrol need to be in place in the country