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Isolation, identification, and characterization of some fungal infectious agents of Cassava in South West Ethiopia

Research Paper (undergraduate) 2016 31 Pages

Biology - Botany

Excerpt

Table of Contents

Abstract:

Table of Contents

Introduction

Materials and Methods
Sample collection
Isolation of fungal pathogens of cassava

Morphological characterization
The effect of culture media on the mycelial growth of the selected fungal isolates
The effect of temperature on the mycelial growth of the selected fungal isolates
The effect of pH on the mycelial growth of the selected fungal isolates
The effect of light on the sporulation of the selected fungal isolates
Pathogenicity tests
Evaluation and testing of antagonistic activities of T. harzianum and T. viride against the selected pathogens
Statistical analysis

Results
Isolation, identification and pathogenicity test of the fungal isolates
Cultural characteristics of the selected isolates
Evaluation and testing of antagonistic activities of T. harzianum and T. viride against the selected pathogens

Discussion

Conclusions

Acknowledgement

Appendix

Isolation, identification, and characterization of some fungal infectious agents of Cassava in South West Ethiopia

Department of Biology, College of Natural Sciences, Addis Ababa University, Addis Ababa, Ethiopia.

Abstract:

Cassava (Manihot esculenta) is a dicotyledonous perennial woody shrub with an edible starchy root, belonging to the botanical family Euphorbiaceous. Apart from its use as human food, cassava products also are popular in international trade under different forms. The crop is economically and nutritionally useful in south western parts of Ethiopia. There is scarcity of information so far on the fungal diseases of cassava in Ethiopia. This study was, therefore, initiated to isolate, identify and characterize some fungal infectious agents of cassava in south western parts of Ethiopia. Samples from organs and the plant tissues were first washed in sterile distilled water and surface sterilized in 1% Sodium hypochloride for one minute and then after in 70% alcohol each for one minute. This was followed by rinsing the plant material in sterile distilled water and allowed to dry on sterile tissue paper. The dried tissues were then cultivated on to PDA and incubated at 250C. Streptomycin sulphate antibiotic 0.05mg/ml was used to avoid the bacterial contamination. Water agar (WA) media was used for sporulation and to have monoconidal isolates of fungi. Monoconidial isolates of the recovered fungi were stored on PDA slants in the refrigerator at 40C for further studies. Eleven isolates were obtained from samples of five locations. However, among these, only six fungal isolates induced pathogenicity on healthy cassava seedlings and leaves. These fungal isolates which grew on glass slide were identified to the genus level by using light microscope as Cephalosporium AAUCF01, Fusarium AAUCF02, Hendersonula AAUCF03, Aspergillus AAUCF04, Penicillum AAUCF05 and Botrytis AAUCF06. The distribution, infection and severity of Fusarium AAUCF02 and Cephalosporium AAUCF01 were more than the other pathogens.

Key words/phrases: - Bio-control, Cassava, Fungal diseases, Pathogenicity, Production

Introduction

Cassava (Manihot esculenta) is a dicotyledonous perennial woody shrub with an edible starchy root, belonging to the botanical family Euphorbiaceous. It belongs to roots and tuber crops that stores edible material in tuber (Howeler, 2003), which belong to class of foods that basically provide energy in the human diet in the form of carbohydrates. Cassava is native to South America, and it is the most widely distributed and cultivated in different parts of the low land tropics. Three continents, Africa, Asia and Latin America produce large amounts of cassava roots. It is the fourth most efficient crop plant, the most widely distributed and cultivated in different parts of the tropics among the tropical root crops (Amsalu Nebiyou, 2003).

Africa is the largest center of production in cassava (Elias and Mikey, 2001). Cassava was introduced to Africa in the latter half of the 16thcentury from Brazil to the West coast of Africa (Jones, 1959; Amsalu Nebiyou, 2003) by Portuguese navigators and later to East Africa through Madagascar and Zanzibar (Charoentrath et al., 2004) and now grows widely in sub-Saharan Africa (Shittu et al., 2007). Although the crop is often regarded primarily as a famine reserve, there has been increasing realization in recent years of its value as a high-yielding source of carbohydrates. Cassava was first introduced into Ethiopia by the British (Amsalu Nebiyou, 2003).

Over 500 million people in the tropical world particularly in Africa depend on cassava as one of the major staple foods. In Asia and Latin America, productions are largely used as raw materials for industries, as animal feed or for export markets. Apart from its use as human food, cassava products also are popular in international trade under different forms such as dried chips, pellets, flour and starch, thus contributing to the economy of exporting countries (Eke et al., 2007). The advantage it has over other crops particularly, in many of the developing world is its outstanding ecological adaptation, low labor requirement, ease of cultivation and high yields. It is also widely cultivated because it can be successfully grown in poor soils, under conditions of marginal rainfall. It has the ability to grow with appreciable yield where many other crops would hardly survive (O’Brien et al., 1991).

It has been estimated that cassava farmers, typically resource-poor farmers, lose 48 million tons of fresh root, some 30% of total world production, valued at US$1.4 billion every year to pests, diseases, and post-harvest physiological deterioration (PPD) (FAO 2002). Because of its long cropping cycle, 8-24 months, cassava is exposed to an array of pests, diseases and environmental pressures over a prolonged period of time. Therefore, the use of costly inputs, such as pesticides, over the entire crop cycle is prohibitive and uneconomical for the small or large cassava producer (Cock, 1982). Furthermore, the fact that cassava is most often (traditionally) grown on marginal soils reduces the plant of important growth enhancing nutrients and exposes the crop to additional stresses making it more susceptible to pests attack and plant pathogens leading to more severe crop losses (Catalayud et al., 2002).

Cassava yield losses up to 80% due to rot diseases have been reported (Theberge, 1985). Among cassava diseases, cassava root rot (Nattrassia mangiferae) and stem rots (Spherostibe repens) are the most important in different parts of West Africa (Wydra and Msikita, 1998; Hillocks and Wydra, 2002). These are among the major constraints of cassava in-ground storage. Root rot, caused by root specific fungi, apart from reducing cassava yield can also reduce the quality of cassava root harvest (Hillocks and Wydra, 2002). A number of fungal diseases of cassava have been reported in some countries of Africa such as republic of Congo, Tanzania, Togo, Nigeria, Uganda and other world parts as follows: Phytophthora drechsleri (Booth, 1978; Theberge, 1985); Sclerotium rolfsii (IITA, 1990); Rosellinia nectarix (Lozano and Booth, 1976; Booth, 1978); Fusarium oxysporum, Botryodiplodia theobromae, Aspergillus niger, Aspergillus flavus, Fusarium solani and Macrophomina phaseolina (Booth, 1978).

Plant pathogens and some insects cause damage and attack on cassava and there by eventually it ends up with a great loss in yield. Fungi are one of the plant pathogens that cause serious diseases of cassava in field conditions (Catalayud et al., 2002). The crop is economically and nutritionally useful in south western parts of Ethiopia. Although research findings have been reported on fungal diseases of cassava in different African countries (Wydra and Msikita, 1998), there is scarcity of information so far on the fungal diseases of cassava in Ethiopia. This study was, therefore, initiated to isolate, identify and characterize some fungal infectious agents of cassava in south western parts of Ethiopia.

Materials and Methods

Sample collection

Diseased cassava samples were collected from different cassava growing areas in order to isolate and evaluate the effect of environmental factors on the pathogens growth. Different parts of the cassava were taken: root, stem and leaf to observe symptoms of the disease. Diseased organs of cassava were sampled from Hawassa/sidama Humbo/Wolayita, W/abaya/Gamogofa, Areka/Wolayita and Jimma zone. The samples were kept in the refrigerator to isolate the fungal pathogens of cassava.

Isolation of fungal pathogens of cassava

Samples from organs and the plant tissues were first washed in sterile distilled water and surface sterilized in 1% Sodium hypochloride for one minute and then after in 70% alcohol each for one minute (Gesier et al., 2005; Summerell et al., 2006). This was followed by rinsing the plant material in sterile distilled water and allowed to dry on sterile tissue paper (Dhingra and Sinclair, 1993; Aneja, 2005; Gesier et al., 2005). The dried tissues were then cultivated on to PDA and incubated at 250C (Roux et al., 2004; Gesier et al., 2005). Streptomycin sulphate antibiotic 0.05mg/ml was used to avoid the bacterial contamination. Water agar (WA) media was used for sporulation and to have monoconidal isolates of fungi. Monoconidial isolates of the recovered fungi were stored on PDA slants in the refrigerator at 40C for further studies (Gesier et al., 2005; Summerell et al., 2006). The isolates were designated as AAUCF01, AAUCF02…AAUCF011.

Morphological characterization

Morphological studies were carried using slide culture technique. Mycelia fragments of the isolates were inoculated on to PDA that was on glass slide and incubated at 250C for 7 days. Afterward, morphological observations were taken based on colony, conidia and conidiophores morphology and other morphological characters as adopted by Barnett and Hunter, (1998).

The effect of culture media on the mycelial growth of the selected fungal isolates

The effect of media was investigated by growing two isolates, AAUCF01 and AAUCF02 on four different media, Potato Dextrose agar (PDA), Potato Sucrose agar (PSA), Malt Extract agar (MEA) and Czapek- Dox agar (CDA). The pH of each medium was adjusted to 3.5. Mycelial fragments were cut off from 7days old culture and inoculated for each medium. Each medium was with three replications and kept in incubator at 250C temperature. After 7 days of incubation, the diameter of the mycelia were measured and recorded.

The effect of temperature on the mycelial growth of the selected fungal isolates

The effect of temperature was investigated for the two isolates on MEA by adjusting the incubator in five levels, 150C, 200C, 250C, 300C and 350C. Mycelial fragments were cut off from 7 days old culture of PDA (Oxoud, pH 5.4 ± 0.2) grown fungal isolates and inoculated on MEA (Oxoud, pH 5.4 ± 0.2). Three replications were prepared for each isolates. After 7days of incubation, the diameter of the fungal mycelial were measured and recorded.

The effect of pH on the mycelial growth of the selected fungal isolates

The effect of pH was studied on potato dextrose broth at 250C temperature. The broth pH was adjusted to five levels, 1.5, 2.5, 3.5, 4.5 and 5.5. There were three replications for each pH value. Mycelial fragments were cut off from 7 days old culture and inoculated on potato dextrose broth, and all these treatments were put on to rotary shaker operating 120 rpm and incubated for 10 days. After 10days of incubation, the mycelial biomass was harvested from the broth by using Whatman No. 42 filter paper and dried in an oven under at 650C for 48 hours. Thereafter, the mycelia dry weight was measured and recorded.

The effect of light on the sporulation of the selected fungal isolates

The effect of light on the sporulation of the fungal isolates was evaluated while growing the isolates under full dark and light for three different durations with the same light intensity (wave length 300-380 nm, near UV and referred as black light) and 250C. Mycelial fragments were cut off from 7days old culture grown on PDA and inoculated on MEA. Thereafter, plates of the fungal cultures were covered with black polyethylene bag to prevent entrance of light (for controls) and the treatments were without cover. Spore counting was done for three times using Heamocytometer.

Pathogenicity tests

The two cassava cultivars, QULLE and KELLO were obtained from Holleta Agricultural Research Center for pathogenicity test with the fungal isolates. Fresh, healthy organs (stem and leaf) from 8-12 month old cassava seedling were washed in running tap water to remove soil and other debris from the surfaces. The organs were surface sterilized in a 1% solution of sodium hypochlorite by immersing them for 1 minute after which they were rinsed with sterile distilled water and left to dry under a laminar flow hood for 3 minutes (Gesier et al., 2005; Summerell et al., 2006). Stems were bored at two edges (upper and lower parts) to a depth of 1cm, using a flame-sterilized 8mm diameter cork borer. A disc of 7 days old PDA culture of each isolate was washed into sterile beakers with sterile distilled water and 1ml of each isolate was inoculated into the hole and sealed with the stems piece removed from the hole. The points of inoculation were sealed with par film to prevent entry of external contaminants as reported by Firdous et al., (2009).

The same procedures were used for the control except that 1ml of sterile distilled water was inoculated into each hole made in the stems. Each inoculated stem was kept in the flasks and incubated at 250C for 7 days in order to observe and examine diseases development. The same procedures were used for leaf inoculation except that sterilized needle was used to make pin micks (holes) on leaf surfaces as adopted by Shah et al., (2009).

In the case of in vivo test, using the above procedures, stem and leafs of cassava varieties were inoculated. Wounds were made on the surface of the root carefully by using sterilized knife. Then, the root was inoculated by drenching 15ml spore suspensions of fungal isolates in to the seedling in polyethylene bags. It was done with three replications for each isolates. And the inoculated seedlings were kept under shade on open air environment for one month. After 7 days of incubation, it was followed up for the manifestation /development of the diseases symptoms as adopted by Shah et al., (2009).

Evaluation and testing of antagonistic activities of T. harzianum and T. viride against the selected pathogens

Dual cultures method

Antagonism of T. viride and T. harzianum against the test pathogens was studied by dual culture technique as adopted by Rama et al. (2000). The selected tested pathogens were grown on PDA for 7 days. The two antagonists were separately grown on PDA for 7 days. Nine millimeter (9mm) disc of the test pathogen culture was transferred to PDA. Similarly, 9mm disc of each antagonist was transferred and put side by side with the test pathogens. They were incubated for 3 days at 250C as the antagonists over grow after 3 days of incubation. The Petri dishes containing the PDA inoculated with the tested pathogen alone served as control. Culture diameter measurements were taken for 3 days consecutively beginning after 24hrs of incubation until the two cultures overlap. The percentage growth inhibition of tested pathogens in the presence of T. viride and T. harzianum were calculated over control. The growth inhibition was calculated by using the formula as adopted by Rama et al. (2000):

Growth in control – Growth in treatment × 100

Growth in control

Statistical analysis

The statistical analysis of growth characteristics of the isolates at different media, temperature and pH, and mean comparisons of the isolates based on different parameters were conducted using one way ANOVA procedures of SPSS statistical analysis software (SPSS institute Inc., Cary, NC) version 13. Differences between treatments were determined by using Duncan Multiple Range Test (DMRT) with (P<0.05).

Results

Isolation, identification and pathogenicity test of the fungal isolates

Eleven isolates were collected from five locations, Hawassa agricultural Center/Sidama, Humbo/Wolayita, Western abaya/Gamogofa, Areka/Wolayita and Jimma zones. However, among these, only six fungal isolates induced pathogenicity on healthy cassava seedlings and leaves (Table2). These fungal isolates which grew on glass slide were identified to the genus level by using light microscope as Cephalosporium AAUCF01, Fusarium AAUCF02, Hendersonula AAUCF03, Aspergillus AAUCF04, Penicillum AAUCF05 and Botrytis AAUCF06.

As shown in Table2, the two cultivars of cassava were infected and colonized by the fungal isolates. Each isolate had different time of the onset of diseases symptom development. The first disease symptom development by Fusarium AAUCF02 was observed after eight days of incubation; small dark circular brown spots appeared on the leaves of QULLE variety which gradually coalesced to form large spots leading to blightening leaves. It was observed that the QULLE variety was more susceptible than KELLO to the test pathogens in terms of the onset of disease symptoms. Re-isolation of the test pathogens from diseased seedlings of cassava revealed that the isolated fungal pathogens were the same as the original identified pathogens of cassava those artificially inoculated on to cassava seedlings. However, some isolates such as Hendersonula AAUCF03 on stem and Penicillum AAUCF05 on leaf didn’t bring apparent diseases symptoms in vitro test.

Cultural characteristics of the selected isolates

The fungal isolates showed different growth diameter on different media at 250C (Table 4). Cephalosporium AAUCF 01 showed maximum mycelial growth (85.3±3.7 mm) on Czapek- Dox agar (CDA) and minimum mycelail growth (68.7±2.9 mm) on Potato Dextrose agar (PDA). The maximum mycelial growth (81.6±2.2 mm) and minimum mycelial growth (78.6±2.3 mm) of Fusarium AAUCF 02 was observed on Malt Extract agar (MEA) and on Potato Dextrose agar respectively. The isolates were grown on PDA to investigate the effect of temperature on mycelial growth after 7days of incubation (Table 5). There was high growth for the two isolates within the temperature range 25-300C. As the temperature went below and above this range, the two isolates showed decrease in growth diameter. They were more sensitive as the temperature went above 300C. The maximum mycelial growth for Cephalosporium AAUCF01 (86.3±3.7mm) was at 300C and it was (82.3±1.5mm) for Fusarium AAUCF02 at 250C. There was minimum growth diameter at 350C for the isolates, Cephalosporium AAUCF01 (21.6±.9 mm) and Fusarium AAUCF02 (30.3±.9 mm). Cephalosporium AAUCF01 was more sensitive than Fusarium AAUCF02 as the temperature went 300C. The two isolates showed different growth at different pH levels (Table 6). There was better growth for both isolates at 3.5 pH. There was reduction in mycelial dry weight beyond this value. The isolates showed more reduction in mycelia dry weight as the pH became below the 3.5 than above it. Fusarium AAUCF02 showed maximum (2.14±.12 gm) and minimum (0.31±.02) mycelial dry weight at pH of 3.5 and 1.5 respectively. Cephalosporium AAUCF01 also showed maximum (2.87±.24 gm) and minimum (0.1±.01 gm) mycelial dry weight at the same pH values as that of Fusarium AAUCF02. Cephalosporium AAUCF01 was more sensitive than Fusarium AAUCF02 as the pH went beyond 3.5 (Table 6). The fungal isolates showed different responses to light on different times of incubation (Table 7). Cephalosporium AAUCF01 showed direct relation with the increase of light duration. Cephalosporium AAUCF01, after 7days of inoculation, showed little sporulation with regard to plates that were covered with black polyethylene bags (dark). However, there was better sporulation on the plates that were exposed to light. There was excellent sporulation after 7days of incubation for Fusarium AAUCF02 in dark where as it was little in light. Culturally, it has no aerial hyphae; colony color, white, smooth in front side of the plate and yellow in back side of the plate.

Evaluation and testing of antagonistic activities of T. harzianum and T. viride against the selected pathogens

The result of in vitro evaluation of antagonistic activities of the two biological agents (T. harzianum and T. viride) showed inhibition of mycelial growth of the test pathogens (Fusarium AAUCF02 and Cephalosporium AAUCF01). T. harzianum had rapid growth over the test pathogens, but it did not show any clear inhibition zone. It showed maximum inhibition (61.5%) and minimum inhibition (20.8%) on Fusarium AAUCF02 after 24hrs and 48hrs of incubation respectively at 250C. T. harzianum inhibition activity was nearly consistent (22-23%) on Cephalosporium AAUCF01 within 72hrs (Table 9). T. viride showed fast growth forming powdery widespread throughout the Petri plates. As that of T. harzianum, it failed to develop inhibition zone, and showed maximum inhibition (52.2%) and minimum inhibition (39.2%) on Fusarium AAUCF02 in the 3rd and 2nd day of incubation, at 250C respectively (Table 9). There was decrease (48.5-43%) in inhibition as the time goes from 1st to 3rd day on Cephalosporium AAUCF01 by T. viride. Average percent inhibition of T. viride was higher than that of T. harzianum for both test fungal isolates of cassava.

Discussion

Eleven isolates were isolated from five different cassava growing areas of South western Ethiopia; of which six isolates were found to incite disease infection on the stem cuttings, leaf and seedlings of QULLE and KELLO cassava cultivars. The isolates were identified by using Illustrated Genera of Imperfect Fungi manual based on their morphological and cultural characteristics (Barnett and Hunter, 1998). They were identified as Cephalosporium AAUCF01, Fusarium AAUCF02, Hendersonula AAUCF03, Aspergillus AAUCF04, Penicillum AAUCF05 and Botrytis AAUCF06.

Even though it was not reported as causative agent of cassava diseases, Cephalosporium gramineum was known to be the causal agent of Cephalosporium stripe of winter wheat (Bockus and Claassen, 1985) and Cephalosporium acremonium, the causal agent of stalk rot and black bundle diseases of maize or Acremonium wilt of sorghum (Bandyopadhyay et al., 1987; Hanlin et al., 1978). Late wilt of maize, caused by the fungus Cephalosporium maydis (Samra et al., 1963), is one of the most important fungal diseases in Egypt. This disease also has been reported from India (Payak et al., 1970; Ward and Bateman, 1999). However, in this study, Cephalosporium AAUCF 01 was identified to the genus level based on morphological and cultural characteristics. The isolate was observed inciting disease symptoms on cassava seedlings and stem cuttings.

Fusarium AAUCF 02 incited wilt on cassava seedlings, and stem cuttings and leaf under incubator at 25 0C. In the same study, Hillocks and Waller, (1997) reported that, in some areas, total crop losses have been attributed to rot diseases. Fusarium species are a significant component of the set of fungi associated with cassava root rot. Yield losses due to root rot average 0.5 to1 ton/ha but losses >3 ton/ha, an equivalent of 15 to 20% yield, often occur. Numerous and diverse species of Fusarium were associated with rotted cassava roots in Nigeria and Cameroon (Bandyopadhyay et al., 2006). Of all diseases caused by Fusarium on cassava, the most important one is the vascular wilt disease caused by formae speciales of Fusarium oxysporum. The Fusarium species that are known to infect a diverse group of plants including crops, ornamentals and trees: F. graminearum, F. culmorum, avenaceum, F. chlamydosporum, and F. verticillioides, which are the most important Fusarium species in central Europe and in large areas in North America and Asia, reduced both crop yield and cereal quality (Gamanya and Sibanda, 2001).

Botrytis cinerea, the pathogen of gray mold disease, causes severe damage on vegetables, ornamentals, fruits, and even some field crops throughout the world. The Botrytis disease is the most common disease of greenhouse-grown crops (Choi et al., 2008). Grey mould rot, caused by Botrytis cinerea is among the most prevalent storage diseases of carrots grown in temperate regions. It was known that some species of Botrytis are plant pathogens with a wide host range that causes heavy yield losses in onion, potato, strawberry, table grapes, and the wine industry (De Curtis et al., 1996). In the other study, Elad et al., (2007) observed that B. cinerea can infect numerous host plants; after infection and death of host tissues, the fungus can survive and sporulate as saprophytes on the necrotic tissue, or produce long-term survival structures, such as sclerotia. In this study, Botrytis AAUCF06 was observed causing infection on cassava crop creating wilt and spots on leaf and canker on stem.

From research works in the other parts of the world, the majority of the species belonging to the genus Aspergillus are reported to be saprophytic in nature. Only a few species including Aspergillus flavus, A. parasiticus and A. niger are considered as weak plant pathogens. These fungi generally infect plant hosts wounded by insects or other agents (Geiser and LoBuglio, 2001). Aspergillus niger causes black mould rot that occurs primarily on roots that are wounded and maintained at high temperature. In this study, it was observed that Aspergillius can infect cassava crop though it was not identified at species level.

In the case of in vitro test, some genera as Penicillum AAUCF05 on leaf and Hendersonula AAUCF03 on stem cuttings didn’t show obvious disease symptoms. This indicates that these genera were organ specific in diseases development on the host. Similarly, Ray and Ravi, (2005) have reported that one of the major constraints of cassava in-ground storage is root rot diseases that is caused by cassava root specific fungi reducing the quantity and quality of cassava root harvest. Although Penicillium is a common postharvest fungus, most pathogenic infections occur preharvestly during fruit development. The genus Penicillium includes about 150 species but only a minor fraction of these cause infection to important plant and processed foodstuffs (Pitt and Hocking, 1997). Several species of Penicillium have been indicated to be pathogenic to a number of different plant hosts. These include P. expansum, P. italicum, P. digitatum, P. solitum, P. viridicatum, P. rugulosum and occasionally P. hirsutum (Pitt, 1991). In Benin and in parts of Nigeria, root rot (Nattrassia mangiferae) is reported to be the most important pathogen (Msikita et al., 2005). Nattrassia mangiferae (formerly known as Hendersonula toruloidea) is a well-recognized plant pathogen causing branch wilt, canker and dieback disease of a wide range of trees, and storage rot of tubers of plants such as yam (Punithalingam and Western, 1989). In this study also, Hendersonula AAUCF03 caused disease symptom on cassava seedlings and leaf but not on stem cuttings.

QULLE variety was more susceptible than KELLO in terms of the onset of disease symptom development and that may be due to various host factors and environmental factors that prevailed to pathogens to incite the disease development. The result of in vitro and in vivo evaluation and testing of six test pathogens on cassava varieties indicated that the higher infection was caused by Fusarium AAUCF02 and Cephalosporium AAUCF01. For this study, therefore, only the two pathogens of cassava, Fusarium AAUCF02 and Cephalosporium AAUCF01 were selected based on their pathogenicity, infection and disease development on the host. Fusarium AAUCF02 was isolated from Areka/Wolayita, West Abaya/Gamogofa and Humbo/Wolayita, and Cephalosporium AAUCF01 was isolated from Humbo/Wolayita and West Abaya/Gamogofa zones (Table2). Their infection severity was high on cassava seedlings in green house as well as leaf and stem cuttings that were incubated at 250 C in laboratory under controlled conditions.

The two genera showed variation in growth on different media as substrate is one of the growth factors. Cephalosporium AAUCF01 got maximum growth (85.3±3.7 mm) on Czapex Docks agar (CDA) and minimum growth (68.7±2.9 mm) on Potato Dextrose agar (PSA). Fusarium AAUCF02 maximum growth (81.6±2.2 mm) was on Malt Extract agar (MEA) minimum growth (78.6±2.3 mm) was on Potato Dextrose agar. The range of diameter was larger on different culture media for Cephalosporium AAUCF01. This showed that it was more sensitive than Fusarium AAUCF02 for culture media composition.

As growth factor, temperature affected mycelia growth. Their better mycelial growth was managed in the temperature range 250C-300C.There was maximum growth for Cephalosporium AAUCF01 (86.3±3.7mm) at 300C temperature. Fusarium AAUCF02 showed maximum mycelial growth (82.3±1.5mm) at 250C. This is in line with the conclusions of Nelson et al., (1983) that 25 0C is suitable for growth of Fusarium species. In general, Cephalosporium AAUCF01 was more sensitive than Fusarium AAUCF02 to wards change in environmental factors and culture media. The genera showed better growth at pH of 3.5- 4.5. Mycelial dry weight showed more reduction when the pH value become below the range than above it. Fusarium AAUF02 showed maximum growth at 4.5 pH. Similarly, Negash Hailu, (2007) reported that the optimum pH for some isolates Fusarium was 4.5. Light affected sporulation of the genera. Fusarium AAUCF02 produced more spores under full dark than light. This indicates that light hindered sporulation for this fungus. Cephalosporium AAUCF01 produced more spores as the light duration increases.

In vitro evaluation of T. harzianum and T. viride indicated that they occupied all the spaces and competed for nutrients thereby hindered the growth of Fusarium AAUCF02 and Cephalosporium AAUCF01. The absence of inhibition zone indicated that the antagonistic mechanism of these species is through competition for spaces and nutrients rather than forming inhibition zone. Similarly, Negash Hailu, (2007) observed that the overgrowth of T. harzianum and T. viride against Fusarium xylarioides. In this study, relatively T. viride exhibited higher percent of inhibition than T. harzianum and this is correlated with the report that T. viride produced antifungal properties that can able to inhibit the plant pathogens (Lin et al., 1994).

Conclusions

In this study, six fungal genera of cassava diseases in Southwest of Ethiopia were reported for the first time. The distribution, infection and severity of Fusarium AAUCF02 and Cephalosporium AAUCF01 was more than the other pathogens. In general, Cephalosporium AAUCF01 was more sensitive than Fusarium AAUCF02 to wards change in environmental factors and cultural media. Each genus had different time for the appearance of diseases symptom. Except Penicillum AAUCF05 and Hendersonula AAUCF03, all genera were causative agents of systematic diseases. T. viride exhibited higher percent of inhibition than T. harzianum on both test pathogens. From this, it is possible to conclude that T. viride is more effective than T. harzianum in controlling cassava diseases caused by Fusarium AAUCF02 and Cephalosporium AAUCF01.

Acknowledgement

The Authors are thankful to all staff members of Holleta Agricultural Research Center for giving cassava seedlings to conduct pathogenicity test. We would like to thank Biology department, AAU for the provision of transport service while travelling to Holleta to bring cassava seedlings. Finally, We are very much grateful to Ms. Nigate Zewiedu, Mycology Lab. Assistant at AAU for her kindly assistance during experimental works.

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Appendix

Table 2. Identification and pathogenicity test confirmation of the fungal isolates

illustration not visible in this excerpt

Key:- “***”, found in three sites; “**”, found in two sites; ++_S, no diseases symptoms on stem; ++_L, no diseases symptoms on leaf and +++, shows diseases symptoms in all cases on both QULLE and KELLO cultivars.

Table 3. Microscopic observation of the selected isolates

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Table 4. The effect of culture media on mycelial growth of Cephalosporium AAUCF 01 and Fusarium AAUCF 02 at 250C, after 5days of incubation (mm)

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* Values mean ± standard error of three replicate; values followed by the same letter are not significantly different (P<0.05).

Table 5. The effect of temperature on mycelial growth of Cephalosporium AAUCF 01 and Fusarium AAUCF02 on MEA after 5days of incubation (mm)

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* Values mean ± standard error of three replicate; values followed by the letter are not significantly different (P<0.05).

Table 6. The effect of pH on mycelia dry weight of Cephalosporium AAUCF01 and Fusarium AAUCF02 after 10days of growth on Potato Sucrose Broth (PSB) (gm) at 250C

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*Values mean ± standard error of three replicate; values followed by the same letter are not significantly different (P<0.05).

Table 7. The effect of light on sporulation of Cephalosporium AAUCF01 and Fusarium AAUCF02 after 7th day, 10th day and 14th day of incubation at 250C.

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D- dark; L- light

Table 8. Cultural characteristics of Fusarium AAUCF02 and Cephalosporium AAUCF01 on MEA after 7days of incubation at 250C

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Key: - -: aerial hyphae absent; +: presence of aerial hyphae

Table 9. Antagonistic effect of T. harzianum and T. viride against Fusarium AAUCF02 and Cephalosporium AAUCF01 using dual cultures technique on PDA

illustration not visible in this excerpt

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2016
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Cassava bio-control fungal diseases pathogenicity

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Title: Isolation, identification, and characterization of some fungal infectious agents of Cassava in South West Ethiopia