DIVERSITY OF PATHOGENIC FUNGI ON PLANTATION FORESTS OF NORTH AND NORTH-WEST ETHIOPIA

Forest plantations in Ethiopia are mainly exotic genera of Eucalyptus, Cupressus, Casuarina, Pinus and native Juniperus species. Plantations species have suffered at varying degrees of attack by disease causing agents, particularly Amhara region is among regions with plantation forests that have in recent years been subjected to attack by diseases in Ethiopia. Plantation trees in commercial stands, farmlands and woodlots were surveyed for diseases symptoms in 20 selected areas of Amhara and Tigray from May to June 2016.Leaf blight, leaf spot, tip blight and stem canker were the most common symptoms appeared during the survey period with leaf spot and stem canker the most prevalent. Tree samples showing clear disease symptoms were collected, surface sterilized, cultured and morphologically characterized for pathogen identification. A total of 42 isolates of fungi colonies were identified from samples collected of 20 localities. Morphological characterization of fungal isolates reveals, six fungal genera belonging to Alternaria, Dioplodia, Pestalotiopsis, Curvularia, Phoma, and Penicillium were the cause of the symptoms of the symptoms of the observed disease. Among the isolates 14 (33.3%) were Alternaria species, 15(37.7%) were Phoma species, and the remaining 13 isolates were Diplodia3(7.2%), Pestalopsis7(16.7%), Curvularia2(4.7%) and Penicillium1 (2. 4%).Based on the findings of the study Phoma lingam, Phoma glomerata, Alternaria alternata, genera of Curvularia, Pestalotiopsis, Penicillium, and Diplodia were found to be the cause of diseases of the tree plantations. Phoma and Alternaria species were the most prevalent isolates, showing a majority of symptoms observed on plantations were due to their co-infection. The pathogenicity test result of the research also confirms fungal isolates were the cause of the symptoms of the disease observed. The findings of this research enable to study and design appropriate management options for the future prevention and control of the diseases especially when there is prolonged environmental stress in the country.


INTRODUCTION
Plantation forests are cultivated forest ecosystems established by planting or seeding using introduced or indigenous species in the process of afforestation and reforestation, primarily for wood biomass production, it coversabout5% of the global forest with Pinus and Eucalyptus species the most commonly used in the world (FAO, 2001). The total area of plantation forests in Ethiopia is estimated to be 972,000-190400 ha for the supply of largest volume of wood products used in the construction sector, the biomass fuel consumed in the country, satisfy household demands for wood and additional household incomes (Lemenih and Kassa, 2014). Yirdaw (2002) showed that forest plantations in Ethiopia are mainly exotic genera of Eucalyptus, Cupressus, Casuarina, Pinus, and native Juniperus species. Majority of forest plantation are located in Oromia, Amhara, the Southern Nations and Nationalities Peoples Region, and Tigray region (Bekele, 2011). According to the study of Jenbere et al. (2011) high rate of plantations compared to other farm, enterprises leads to the extent of conversion croplands and grazing fields to Eucalyptus woodlots in Amhara region. An income from plantations like Eucalyptus tree sales contributes on average up to 25% of total household annual cash income and for poor households up to 72% of the total annual cash income which is the largest non-agricultural source of household income in the country (Jegger, 2003).
Successful forest plantations require species well matched with sites, improvement of genetic stock, control and related operations that enhance tree quality and stand growth. Currently, plantations are more at risk from diseases due to pathogens than natural forests (Evans, 2000). Among the wide variety of pathogens that cause plant diseases, fungi are important stressors that affect tree health. The magnitude of fungal diversity is estimated to be 1.5 million species, with only 5% of species were described. Available evidence also indicates that fungal diversity in the tropics is richer than others (Berrin et al., 2012). Large numbers of fungi species are plant pathogens causing about 70% of plant disease. Fungal plant pathogens, if not controlled one way or the other, can have a devastating effect on biodiversity, forest structure and dynamics, commercial plantations, agroforestry and urban environments. The impacts of fungal pathogens on plantation forestry species can be more severe with increased movement of humans and plant products around the world as it facilitates distribution of diseases causing agents to new areas of the world (Chimwamurombe, 2016). Forest plantations of Ethiopia are at varying degrees of attack by disease causing agents, particularly that of Amhara region are among that have in recent years been subjected to attack by diseases. Study of Gbadegesin et al. (1999) reveals that leaf disease of Eucalyptus, cypress, shoot blight and dieback of Pine are common diseases in the region, with most of the diseases observed are expected to be from nursery sites. Fungi species can associate a host plant as phylloplane, saprobes, endophytes, mycorrhizal, parasites or commensals (Tang, 2003). Plant disease complexes can also involve association of more than one pathogenic fungus in a host as in brown apical necrosis of walnut fruit where numerous plant pathogenic fungi Fusarium, Alternaria, Cladosporium, Colletotrichum, Pestalotiopsis and Phomopsis involved (Lamichhane and Venturi, 2015;Lee, 2003). Most fungal species that cause diseases such as leaf spot, leaf blight and stem canker show overlapping symptoms of small, scattered, circular to oval dead areas in the leaves; usually tan, dark brown, yellow, gray, purple, or black with some spots rose, shiny, and coal black forming ragged holes with marked light and dark concentric zones (Figure 1). The objective of this study was to investigate type, diversity, and distribution of pathogenic fungi associated with plantation forest trees showing diseases symptoms in Amhara and Tigray region, North and North West of Ethiopia. Material and methods Study areas, Sampling and Sampling techniques Study areas: Amhara Region is located between 8°45'N and 13°45'N latitude and 35°46'E and 40°25'E longitude in North West Ethiopia with annual mean minimum and maximum temperatures between 15°C and 21°C and the average annual rainfall of 1194 in mm. Tigray forms the northernmost reaches of Ethiopia, and is located between 36 degrees and 40 degrees east longitude, north-south extent spans12 and a half degrees to 15 degrees north with average annual rainfall between 450-980 in mm, and the annual minimum and maximum mean temperature of the region is between 9.86°C and 24.9°C (Bewket, 2009;Taye et al., 2013;Ayalew et al., 2012). Sampling and Sampling techniques: Plantation trees in commercial stands, farmlands and woodlots were surveyed in the selected areas of Amhara and Tigray regions in moist season from May to June 2016 for disease symptoms. Random sampling was used in the collection of samples from 20 plantation sites, based on Gbadegesin et al. (1999). The severity of the problems in the areas as pointed out by Regional, Zonal and District Forestry and protection staffs. Identification and morphological characterization of fungal pathogens: Leaves, pieces of bark, twigs and segments of stems showing disease symptoms were collected and processed for identification of causal fungi from plant tissues exhibiting clear symptoms. Infected tissues aseptically cut into small pieces (2-5 mm squares) along with adjacent small unaffected tissue were surface sterilized transferring to sterile Petri dishes containing tap water,90% ethanol solution and distilled water for 30-60s. The sterilized pieces were transferred to Petri dishes containing potato dextrose agar (PDA) in a hood and incubated at room temperature (25-30°C) for 5-7 days and examined daily for the growth. Morphological studies of cultures isolate on PDA were conducted following the methods described by (Boerema et al., 2004). Micromorphological descriptions for 42 fungal culture were carried out from mature conidiomata and conidia using slides mounted in water (Aveskamp et al., 2010;Chen et al., 2015). Slides were prepared to make detailed observations of the morphological features, size, and shape, colors of conidiomata, pyinida, conidia and patterns of fungal growth in vitro using a compound microscope. Colony colors on the surface and reverse of inoculated Petri plates were assessed according to the color charts of (Rayner 1970). Fungal cultures were identified at genus and species level based on observed macroscopic and microscopic characteristics (Khan et al., 2015;Ngobisa et al., 2015;Raymond et al., 2000;Saju et al., 2011). Pathogenicity trials: Pathogenicity of fungal isolates was tested using seedlings of the respective host plants raised in the nursery sites of Central Ethiopian Environmental and forest research center(CEE-FRC). As it is not practical to test all the fungi isolated to their respective host plants to establish the pathogenic status, only selected fungi (rarely encountered ones and weak pathogens) were screened. Pathogenicity of the fungal isolates to the respective hosts was tested by using 3-6 month-old seedlings and spraying conidial suspension (2 x 103 conidia/ml of sterile water) of the respective fungus. Three to five leaves of seedlings of the respective host plants were inoculated and incubated in a humidity chamber (>90% R.H, 26 ± 2 0 C with 12h dark and light period). Disease symptoms developed were recorded and fungi were re-isolated from the diseased host tissues and pathogenicity of the respective fungal species confirmed (Xue et al., 2004). Data Analysis: The collected data were summarized, ranked and expressed using simple descriptive statistics such as percentages and graphs. Survey data of Morphocultural characters. The relative prevalence of each pathogenic fungal species with respect to localization and others were analyzed using SAS Ver. 9 procedures at probability level, p=0.05 (SAS Institute Inc., 2002). RESULTS Symptomology of disease: Symptoms of several diseases were observed on Eucalyptus, Cupressus, and Juniperus and Chordia species at study sites. Most of the study sites were dominated by Eucalyptus plantations, particularly Eucalyptus globulus in the high land areas. The most common symptoms appearing during the survey were leaf blight, leaf spot and stem canker typical of those caused by Phoma, Alternaria, Curvularia and Pestalotiopsis sp. on Eucalyptus and Chordia spp. and tip blight symptoms similar to infection by the Diplodia sp. (Figure 1) on Juniperus procera and Cupressus lusitanica of plantations. Leaves and stems were associated with brown to black spots, round to irregular-shaped. Leaf spots were circular or irregular in shape separated or aggregated and often located at the margins with brown, pale brown to grey coloration while stem canker is observed with elongated, greyish, hell brown to the dark brown border between discolored tissues lesions typically on Eucalyptus globules as shown in figure 1.

Morphological characterization of fungal isolates:
A total of 42 isolates of fungal colonies were identified from 20 localities (Table 1). Colony textures of the isolates on PDA appeared as appressed with sparse aerial mycelium with raised and slightly dense aerial mycelium, or floccose with raised and dense aerial mycelium. Colony colors of the isolates were observed white, gray, and black, brown, green to dark green and pink (Figure 2). Mean colony diameter of the fungal isolates on the PDA were found insignificant with ranges from 4.48cm to 6.25cm on 9cm Petri plate within seven days of incubation at 25-30°C (Table 2).  Morphological feature of fungal isolates reveals, six fungal genera belonging to Alternaria, Diplodia, Pestalotiopsis, Curvularia, Phoma, and Penicillium were the cause for the diseases symptom observed in the study areas ( Figure 2). Among the isolates 14 (33.3%) were Alternaria species, identified as A. alternata, 15 (37.7%) were Phoma species, identified as Phoma lingam and Phoma glomerata while remaining 13 isolates belongs to genera Diplodia (7.2%), Pestalotiopsis (16.7%), Curvularia (4.7%) and Penicillium (2.4%). Results of the study also show, species of Phoma and Alternaria were the most frequently isolated and found to be pathogens for the majority of stem canker and leaf spot diseases symptoms observed in the survey areas ( Figure 3). Pathogenicity result of fungal pathogens revealed typical symptoms of the disease appeared after 10 days of inoculation on host plants confirming the causal agents for the diseases on the plantations.

DISCUSSION
Morphological characterization result of fungal isolates indicates, six fungal genera belonging to Alternaria, Diplodia, Pestalotiopsis, Curvularia, Phoma, and Penicillium were identified. Tang (2003) stated that several genera of fungi, notably Pestalotiopsis, Diplodia, Alternaria, and Phoma are responsible for infections on plants; they are latent opportunists (endophytes) that colonize and cause asymptomatic infections in healthy plant tissues. In the current survey, it was found that Phoma and Alternaria were the most abundant. They were found in 29out of 42 fungal isolates studied. According to the findings of Zimowska (2011) Fungi of the genus, Phoma are at present cosmopolitan in respect of geography consisting of a large number of species in varied ecological niches with the majority are pathogenic species known infecting plant that is economically important. Boerema et al. (2004) also indicated that these species are found in association with symptoms of blight, leaf spots; fruit rot and stem canker throughout the world. The study of Aveskamp et al. (2008) also strengthen this idea in that, Phoma diseases are most prevalent in cool, wet weather, light and frequent rains, fog or heavy dews, high humidity, and crowded or shady plantings with infection occurring any time from June to August following temporary periods of cool, wet weather. Regarding Alternaria and Pestalotiopsis species, Fernández et al. (2015) reported that they are aerial plant pathogens, infecting plant tissues usually facilitated by injuries and simultaneous isolation of the two species from symptomatic leaves, branches, and a fruit is very common. According to Keith et al. (2006), different species of Pestalopsis caused leaf spots, needle blight, tip blight, and a gray blight on a range of hardy ornamentals plants.
Diplodia species are widely distributed opportunistic pathogens of conifers like Pinus spp. worldwide (Bihon et al., 2011;Hanso and Drenkhan, 2009). Curvulariaspp.is among fungal plant pathogens that cause leaf spot diseases associated with dark brown pinpoints in most parts of the leaves forming dark brown lesions surrounded by yellowish halos, which finally became diffused leaf blight (Sunpapao et al.,2014). Penicillium is among fast-colonizing opportunistic fungi characterized by the fast establishment on wounds or susceptible regions of leaf, stem and other parts of plants (Tang, 2003). According to Ezekiel et al. (2008), Aspergillus species, Penicillium species, Curvularia species Alternaria species, and Phoma species are seed fungal pathogens, this indicates they can possibly cause diseases at nursery level and further at plantation site if there is no appropriate fungal pathogen management system for seeds at storage and seedling at nursery level. This study helped to have overall pictures of fungal diversity on plantation forests dominated by Eucalyptus species in Amhara and Tigray regions, North and North-West Ethiopia with associated symptoms. In the study areas, exotic host species are found more vulnerable to fungal pathogens than indigenous one. Leaf spot and stem canker are the most prevalent symptoms on plantations. Phoma lingam, Phoma glomerata, Alternaria Alternata and genera of Curvularia, Pestalotiopsis, and Penicillium including Diplodia were found to be the cause for symptoms observed on plantations. Among the isolates Phoma and Alternaria genera were observed the most prevalent, showing a majority of leaf spot and stem canker symptoms on plantation trees were due to these fungal pathogens, which is also an indication for leaf spot diseases to be due to co-infection of more than one fungal species on a host.

CONCLUSION
The results of Survey of Plantation Forests Plantation trees in commercial stands, farmlands, and woodlots of Amhara and Tigray regions indicates existing diversities of fungal species which can possibly cause diseases when there is prolonged environmental stress. Based on the findings of the study Phoma lingam, Phoma glomerata, Alternaria alternata, genera of Curvularia, Pestalotiopsis, Penicillium, and Diplodia were found to be the cause for diseases symptoms observed on the tree plantations. Phoma and Alternaria species were the most prevalent isolates, showing a majority of symptoms observed on plantations were due to their co-infection.