Abstract: Background and Objective: Eucalyptus globulus species are exotic plantations in Ethiopia covering one-third of the total plantation area of the country mainly for construction and resources. Inspite of this importance the success of growth and development of E. globulus plantations is currently influenced by fungal pathogens causing leaf spot and stem canker diseases. The main objective of this study was to investigate the diversity and distribution of fungal species associated with plantations causing leaf spot and stem canker diseases, so that management options to be studied for effective control and prevention of the diseases. Methodology: Twelve plantation sites were purposivly sampled and surveyed in Amhara and Tigray regions of Ethiopia from May-June, 2016 for disease symptoms. Samples with clear diseases symptoms were collected cut to desired size, surface sterilized and cultured on the potato dextrose agar media for morphological study. Twenty pure cultures of fungi colonies were isolated and morphologically characterized to genus and species level for identification. The collected data were summarized, ranked and expressed using simple descriptive statistics of SPSS version 16.0 and SAS Version 9.0 procedures. Results: Based on the results, among the isolates 60% (12 out of 20) isolates were with long chains or solitary, unicellular or multicellular, alternarioid chlamydospores while the rest of isolates were with milky conidia, two oil droplets inside, oozing in characteristic shape from dark pyinida and without chlamydospores. The morphological characteristics of the isolates were found to be genus Phoma and the species were consistent with Phoma glomerata and Phoma lingam, respectively. The mean colony diameters of the isolates were range from 7-8 cm and 7.3-8 cm for Phoma lingam and Phoma glomerata, respectively. There is no significance difference in mean colony diameter among the isolates. Conclusion: The result of this study demonstrated that Phoma glomerata and Phoma lingam were the cause for leaf spot and stem canker diseases of E. globulus tree plantations in high lands of Ethiopia, which is important in the study of management options for control and prevention of the diseases in the country.
INTRODUCTION
Eucalypts of the Family Myrtaceae (subfamily Myrtoideae) is among the dominant genuses of flowering plants in the world, consisting about 800 species1. Planting of Eucalypts in Ethiopia has historically dating back to the late 1800s2,3. Eucalyptus globulus species are among exotic plantation in Ethiopia covering about one-third of the total plantation area of the country mainly for the benefit of construction, fuel, poles and an important resource for subsistence farmers4. Introduction of eucalypts plantations to the country was mainly due to deforestation of native forests for farm lands and construction materials5. It has been shown by Mengist6 that introduction and distribution of fast growing exotic tree species such as Eucalyptus globules in the country was to minimize the problem of wood biomass crisis and construction materials. As already pointed out by Alemu7, Eucalyptus plantations has become an alternative income source and employment opportunity in the cash crop devoid high land areas of Ethiopia. Among Eucalyptus species, Eucalyptus globules were found the most effective in terms of survival and growth8.
According to Bekelle9 and other researchers out of the total area of small areas of plantation, about 35% are dominated by E. globulus for variety of products including, leaves and small branches for fuel, poles and posts for house building and sources of income in Amhara region of Ethiopia.
Inspite of this importance the success of E. globulus plantations is currently influenced by several factors in Ethiopia. Among the factors that challenge growth and development of these tree species are fungal pathogens mainly from Ascomycota. Approximately about 700 species of fungi are associated with Eucalyptus leaves and stems10. Cylindrocladium spp. Mycosphaerella spp., Phaeoseptoria eucalypti spp., Pestalotiopsis spp. and Pseudocercospora eucalyptorum spps are among important fungi that cause diseases to Eucalyptus spp.4,11.
Fungi of the genus Phoma are at present cosmopolitan in respect of geography consisting of a large number of species in varied ecological niches. From among 3000 taxa described so far, 110 are pathogenic species often infecting plant that are important from the economic point of view10. Phoma sp. are frequently found in association with symptoms of blight, leaf spots; fruit rot and stem canker throughout the world12. The disease is most prevalent in temporary periods of cool and wet weather associated with light and frequent rains13.
Most leaf spot diseases and stem canker develop as small, scattered, circular to oval dead areas in the leaves; usually tan, dark brown, yellow, gray, purple, or black. Some spots are raised, shiny and coal black, others may drop out leaving ragged holes; some are marked with light and dark concentric zones (Fig. 1). Numerous spots develop yellow, purple, red or reddish brown to black margins; and later, in damp weather, increase in size and number can merge into large, angular to irregular dead areas. Dark areas and speck-sized, fungus-fruiting bodies (known as pycnidia, acervuli and perithecia) commonly form the dead tissues of many older spots (Fig. 2). Heavily infected leaves may turn yellow to brown, wither and drop early, weakening the tree. Occasionally, some leaf spotting fungi deform or kill flowers, buds, fruits, twigs, or even small branches (Fig. 1). In Ethiopia, research on tree diseases in plantation and natural forests is at infant stage, stress from climate change and other factors brought loss of plantation trees due to diseases and this is increasing from time to time in the country. To overcome this problem, research on type, prevalence and management of plantation diseases is very important. The objective of this research was to investigate the diversity and distribution of fungal species associated with plantation of E. globulus causing leaf spot and stem canker diseases in North and North West Ethiopia. The result of this study will help at large in the study of effective diseases management options for effective control and prevention of E. globules diseases. This study also aims to contribute base line findings and information that initiate researchers for further tree disease research in the country.
| Fig. 1: | E. globulus plantations in the field showing stems canker and leaves spot symptoms |
MATERIALS 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 and 21°C and the average annual rain fall of 1194 in mm12. Tigray forms the northernmost reaches of Ethiopia and is located between 36 and 40° east longitude, north-south extent spans 12 and a half degrees to 15° north14. The average annual rainfall between 450-980 in mm and the annual minimum and maximum mean temperature of the region is between 9.86 and 24.9°C15.
Sampling and sampling techniques: Eucalyptus trees in commercial stands, farm lands and woodlots were surveyed in the selected high land areas of Amhara and Tigray regions in moist season from May-June, 2016 for presence of disease symptoms. The survey and sampling procedure were according to the procedures of Banito et al.16 with modification. Twelve plantation sites were purposively surveyed based on severity of the problems in the areas, from each plantation sites 3-4 plants were randomly selected by walking in an "X" fashion for collection of samples. At least four to five leaves, twigs and stems were collected from each plant within the plantations for identification.
Identification and morphological characterization of fungal pathogens: Leaves, twigs and segments of stems showing disease symptoms were collected and separately placed in brown paper bags, which were sealed in larger plastic bags to retain moisture, until isolations were done. Sample culturing, identification and morphological study were done according to the procedures of Aveskamp et al.13, Boerema et al.17 and De Castro Silva18. After washing the tissues thoroughly with sterile water, the causal fungi were isolated from plant tissues exhibiting clear symptoms. The infected tissues along with adjacent small unaffected tissue was cut into small pieces (2-5 mm2) and by using flame-sterilized forceps, they were transferred to sterile Petri dishes containing tap water, then to 90% ethanol solution and then to distilled water for surface sterilization of plant tissues for 30-60 sec. Three sterilized pieces were aseptically transferred to Petri dishes of 9 cm diameter containing Potato Dextrose Agar (PDA) in triplicate and incubated at room temperature (25-30°C) for 5-7 days and examined daily for the growth of the organism. The cultures were incubated during the first week in a thermostat without access of light at the temperature of 22°C and for the next 13 h in UV light and 11 h in darkness. Samples that could not be processed immediately were kept in cool dry conditions or in a refrigerator at ~4°C.
Morphological studies of cultures isolate on PDA were conducted following the methods described by Boerema et al.17. Colony diameters were measured after 7 days converted to mean colony diameter and colony morphologies were determined after 7-14 days of incubation. Colony colours on the surface and reverse of inoculated Petri dishes were assessed according to the colour charts of Rayner19. Micromorphological descriptions for 20 Phoma spp., culture of relevant features were carried out from mature conidiomata and conidia slides mounted in water13,20. Slides were prepared to make detailed observations of the morphological features, size, shape, colors of conidiomata, pyinida, conidia and chlamydospores and patterns of fungal growth in vitro using a compound microscope. The keys considering the current principles of the taxonomy of fungi from genus Phoma were used for identification21. Fungal cultures were identified at genus and species level on basis of macroscopic characteristics like colony morphology, color, texture, shape and appearance and microscopic characteristics like conidia shape, hyphae color, septation, concentric zone, pigmentation, fruiting bodies or any other visible structures22-25.
Pathogenesity tests using detached leaf technique: Mycelia disks from each Phoma isolate were used to inoculate leaves old field-grown E. globules. New leaves free from leaf spot symptom, were collected, washed and surface sterilized with 70% ethanol, 0.5% sodium hypochlorite and rinsed with sterile distilled water, prior to inoculation and sprayed with the spore solution of evaluated fungal isolates. Controls were sprayed with sterile water free of fungal spores. After inoculation, leaves were placed in humid plastic bags and kept at room temperature. Symptoms were recorded 5 days after inoculation and re-isolation, was made from all resulting lesions according to Badillo-Vargas et al.26 and Torres-Calzada et al.27.
Data analysis: The collected data were summarized, ranked and expressed using simple descriptive statistics such as percentages and graphs. Survey data of morpho-cultural characters, relative prevalence of each pathogenic fungal species with respect to localization and others were analyzed using SAS procedure Version9.0 at probability level, p = 0.0528.
RESULTS
Symptoms: On site observation of leaf spot and stem canker symptoms on E. globulus plantation was made in May and June, 2016. Brown to black spots, round to irregular-shaped, occurs on leaves and stems. Leaf spots were circular or irregular in shape separated or aggregated and often located at the margins with brown, pale brown to grayer coloration while stem canker is associated with elongated, grayish, hell brown to dark brown border between discolored tissues lesions (Fig. 1).
Morphological characterization of fungal isolates: A total of 20 isolate of fungi colonies consisting of Phoma species were identified and further characterized (Fig. 2).
The mycelium develops slowly, forming round to wedge-shaped colonies on PDA medium. After 7 days, pink to grey, green to dark green and white to grey colour mycelia sectorially or in concentric zones and round, dark brown to black Pycnidia was observed (Fig. 3). The mean colony diameter was found to be range from 7-8 cm and 7.3-8 cm for Phoma lingam and Phoma glomerata, respectively after fourteen days. The mean standard errors of the isolates show that there is no significance difference in mean colony diameter among the isolates on the PDA (Table 1).
Colony textures were recorded as either appressed with sparse aerial mycelium, flocculose with raised and slightly dense aerial mycelium, or floccose with raised and dense aerial mycelium (Fig. 3). Colony colors were observed as white, gray and pink. Colony shape was either uniform with smooth edges, irregular with rough edges or banded with sectors consisting of thin expansive mycelium. Morphological variations were observed among the isolates with specific morphological or cultural traits (Fig. 3).
Conidia were single-celled, hyaline, short cylindrical, with oil drops on both tips while multicellular chlamydospores of alternarioid shapes were also observed (Fig. 4).
Pathogenesity tests conducted under laboratory conditions showed that all Phoma isolates evaluated were pathogenic to E. globulus leaves brought from nursery. Gray, brown to black spots on leaves was observed seven to twelve days after inoculation. Control plants did not show symptom development after four weeks of periodical observations.
DISCUSSION
The survey depicted that leaf spot and stem canker diseases were found the most prevalent on E. globulus plantations in the highland fields of Ethiopia. The findings of this study revealed that the diseases were due to fungal pathogens belonging to genera of Phoma.
| Fig. 2: | Number and types of Phoma species isolated from Amhara and Tigraye regions |
| Fig. 3(a-c): | Morphological variabilities among Phoma spp., isolate from stems and leaves of Eucalyptus globulus: (a, b) Phoma glomerata front and reverse side and (c, d) Phoma lingam after 7 days of incubation |
| Fig. 4(a-f): | Phoma spp., isolates microscopic structure, (a) Phoma glomerata Pycnidia (b, c) Phoma glomerata alternarioid chlamydospores (d), Phoma glomerata free conidia (e, f), Phoma lingam Pycnidia with characteristic shape of oozing spores and free spores |
The morphological characteristics of pure fungal culture were found to be varies in texture, color and margin. Among the isolates, majority were apprised in texture while the rest were floccules and floccus (Fig. 2). In terms of colony color, isolates were range from white to grey, light pink to white and dark green to black on PDA but, colony margins of most isolates were observed regular, this result is consistent with the findings of Aghapour et al.21 showing variation among Phoma spp., colonies within and among groups, depending on culture medium, substrate and environmental conditions. Based on observed morphology, circular, white to pink and, dark green to grey color with long chains or solitary, unicellular or multicellular alternarioid chlamydospores. The morphological characteristics were consistent with morphological descriptions of Zhao and Kang10, showing the Phoma species to be Phoma glomerata. The relative high prevalence of the diseases in the area is attributed to the production of conidia with a relatively fast growth rate during suitable weather condition enabling the fungi to invade large number of hosts within short period of time, which is in line with the study of Banito et al.16. The rest of pure culture isolates with milky white to gray color mycelia, associated with free conidia oozing in characteristic shape from dark pyinida, oval shaped, unicellular and each with two oil like droplets inside, without chlamydospores were identified as Phoma lingam, which agrees with research findings of Aveskamp et al.13 and Boerema et al.17. According to the findings from morphological study Phoma glomerata and Phoma lingam are found to be the cause for leaf spot and stem canker of E. globulus plantations which is again in line with the study of others such as Aghapour et al.21, Chen et al.29 and Hammoudi et al.30. The results of this study also confirm, the cosmopolitan nature of Phoma spp., showing majority of plants being infected worldwide as stated by Aveskamp et al.13 and Boerema et al.17. This nature of Phoma spps is also stated by Aveskamp et al.13 as P. eupyrena, P. exigua, P. glomerata, P. herbarum and P. macrostoma are the most frequent occurring Phoma species worldwide, irrespective of the climatic conditions. This study is an evidence for Phoma spp., to be a cause for stem and leaf disease in E. globulus plantations which is also indicated in the study of Sivasithamparam et al.31 and Yuan and Mohammed32.
| Table 1: | Morpho-cultural and microscopic characteristics of fungi isolated in the study areas |
| *PDA-Potato Dextrose Agar, *masl-meter above sea level | |
Studies in Zhang et al.33 also supported this finding showing Phoma fungi that cause leaf spots grows along the leaf petioles to the stems and result in stem canker during favorable environmental conditions. Researchers such as Bettucci et al.34 and others also stated that Phoma spp., are among Endophytic fungi colonizing leaves, stem and twigs of E. globulus and even their seedlings, depending on the stress from environmental variables they can possibly be aggressive pathogens that cause severe losses to these plants. The research of Aveskamp et al.35 also indicates, Phoma spp., can cause maximum yield loss usually during nursery stages and cause significant yield loss of the tree plantations when there is prolonged environmental stress. The Pathogenecity study of the Phoma isolates described showed that they are pathogenic to sprayed E. globulus leaves and produced identical symptoms to those observed in the field, confirming the Phoma isolates causal agents for leaf spot disease of E. globulus in Ethiopia, which is also in line with findings of Zimowska36. Beside the absence of molecular analysis and limitation of the study to specific parts of the country due to time and financial related constraints, the findings of this research from morphological study show that Phoma species could be the cause for leaf spot and stem canker diseases of E. globulus in the high land areas of Ethiopia. The findings of this study will be expected to contribute much in the study and application of effective management options with in depth research including other high land areas of the country for effective control and prevention of the diseases.
CONCLUSION AND FUTURE RECOMMENDATION
In conclusion, E. globulus tree plantations are important sources of economy in North and North West Ethiopia. E. globulus production is currently with great challenges in the regions because of pathogen related problems. On the basis of cultural and morphological characteristic of fungal isolates Phoma spp., were the cause for severe leaf spot and stem canker diseases on E. globulus tree plantations in Amhara and Tigray regions of Ethiopia. The pathogens were identified and characterized to genus and species level through morphological characteristic and found to be Phoma glomerata and Phoma lingam. The result of pathogenesity analysis revealed that Phoma fungi isolates were pathogenic to E. globulus plants forming similar symptoms with samples collected from the research sites.
Further long range research is needed for enough data of incidence, prevalence and severity of Phoma fungi on E. globulus plantations. Morphological characteristic method assisted with modern approaches including molecular tools, would provide a better understanding and reliable information of Phoma isolates to species level. Awareness about the spread of this disease to nursery site workers, farmers and plantation owners help the diseases to be controlled and protected at source level. Additional survey of the diseases at nursery sites is recommended for comparison of diseases parameters at plantation and nursery site. Nurseries have to be assessed for diseases symptoms and there has to be appropriate treatments for diseased E. globulus seedlings before distribution to society’s plantation sites.
SIGNIFICANCE STATEMENTS
The researcher identified the fungal pathogens that cause the E. globulus diseases which provide information at large in the study of management options for future control and prevention of the diseases.
ACKNOWLEDGEMENT
The authors thank Ato Abraham Yirgu, Alemu Gezahegn (Dr) and Adane Tesfaye (Dr) of Forest Protection Process research team for provision their kind words, strengths and comments during the field and laboratory works. We also thank Tiruwork Tesfa and Almaz Assefa, laboratory assistants of Forest Protection Process for their valuable assistance during the laboratory phase of the research.