An Optimized Inoculation Method of Fusarium Wilt (Fusarium oxysporum f. Sp. cubense) Causal Agent of Banana Wilt Disease in Ethiopia

Endriyas Gabrekiristos, Mohammed Yesuf and

Getachew Ayana

Greener Journal of Plant Breeding and Crop Science, vol. 6, no. 2, pp. 007-014, May 2018

Ethiopian Institute of Agricultural Research, Melkassa Agricultural Research Center P.O. BOX 436, Adama, Ethiopia.

1. INTRODUCTION

Fusarium wilt is one of the most notorious diseases that affect banana plants. The disease is caused by the soil-borne hyphomycete, Fusarium oxysporum f. sp Cubense (Foc) (Ploetz, 2015). In terms of crop destruction, Fusarium wilt ranked the foremost devastating plant diseases such as wheat rust and potato blight (Carefoot and Sprott 1969). In the period 1890-1960, some 40,000 hectares of the susceptible banana cultivar Gros Michel (grown for export) were destroyed or abandoned in Central and South America and the Caribbean because of race 1 of Foc. Based on  its virulence in specific banana cultivars (Ploetz, 1990,1994), the pathogen can be classified into three races: 1, 2 and 4. Production and productivity of banana is high in Northern and Southern part of Ethiopia, however diseases caused by fungi, bacteria and nematode affect banana plantation (Jones 2000). Among others fusarium wilt and sigatoka distributed  across main banana producing region of the country.  The soil-borne fungus, Fusarium oxysporum is the causal agent of vascular wilt, a disease that affects a large variety of economically important crops worldwide (Ortoneda et al., 2004). Other than Fusarium wilt  burrowing nematode, banana anthracnose, cigar end rot, bacterial wilt and black sigatoka were  important disease of banana in Ethiopia. Fusarium wilt and Black sigatoka diseases were reported in 2014 around Tepi Agricultural Research Centre which is potential in banana production areas.  From assessed area fusarium wilt was sever in Dale woreda (84%), Aleta Chuko (36%) and Samen Bench (36%) (un published ) districts. Use of resistant genotypes is the best control practice for Fusarium wilt (FW) of banana also called Panama disease. Genetic improvement plays an important role in transferring disease resistance alleles to the genetic background of elite genotypes. In addition, field tests are time consuming (3 years) and demanding in terms of manpower and space. Hence, a rapid and reliable method of inoculation is vital for early detection of resistant genotypes.  Several protocol to infect banana with fusarium wilt under green house condition have been reported. The present paper describes the way to produce symptoms of fusarium wilt in banana variety by considering several factors and to optimize efficient protocol for further research on identifying resistant or tolerant variety of released and pipe line banana varieties.

2. MATERIAL AND METHODS

2.1 Study area

The experiments were carried out  at Melkassa Agriculture Research Center (MARC) located at 8⁰24’985 N latitude and 39⁰19’529 E longitudes, with an altitude of 1,550meter above sea level. The annual average maximum temperature is 27°C the minimum average temperature 20°C and minimum relative humidity was 50 percent and the maximum relative humidity was 80 percent.  The investigations were carried out under greenhouse condition.

2.2 Culture media and Inoculum production

Samples with a symptom of fusarium wilt were collected from Melkassa Agricultural Research Centre, Sidamo Zone and brought to MARC plant pathology Laboratory. Samples were washed with bleach and distilled water to remove saprophytic fungi to get pure culture. In this experiment potato dextrose agar (PDA) was used to grow pathogen. After seven days of incubation, pathogens developed from cultured samples were identified based on their conidia shape and other fruiting structures with the help of stereomicroscope and descriptors. From actively growing PDA cultures of fusarium spp, aseptically the spore were placed at the centre of other Petri dish with diameter of 9cm containing media for purifying the pathogen.  Then inoculate plates were incubated in mould incubator with adjusted temperature of 26+2 and relative humidity above 75% for 10 days.  Seventeen day old culture were identified by morphology and conidial shape under stereomicroscope and scarped in to cylinder.  Then the pathogen were  shacked on shaker for twelve hour in order to get uniform conidia. Haemocytometer and hand tally counter were used to adjust suspension.

2.3 Planting materials and Observations

Acclimatized three month old tissue cultured plantlets of banana variety (poyo) which is susceptible to all race of fusarium were used to see the effectiveness of the optimized protocol to inoculate fusarium wilt. Plants  grown in a disease-free environment with no contact with Foc, which may eventually provoke accidental or cross-contamination. Plantlet were 15-25 cm height and shows no nutrient deficiency. Treated and untreated seedlings were managed with optimum  agronomic practice to get normal farmers practice except the pathogen case. Untreated seedlings were isolated from inoculated(treated)  one to minimize cross contamination of test pathogen.

2.4 Optimized protocol to inoculate fusarium wilt  of  banana on banana seedlings

1.   Susceptible variety was selected and multiplied by tissue culture and grown for three month under aseptic condition.

2.   Three months old seedlings were brought to Melkassa Agricultural Research Centre,  plant pathology laboratory and seedlings were removed from the pot and washed neatly with tap water when necessary.

3.   17 day old purified culture were adjusted with  a spore concentration  of 1 X 10⁶ spore/ml and excess roots were   trimmed to favour  pathogen penetration and during all step avoid plant stress  and keep plants with adequate water levels.

4.   Leave the plant root contact with suspension adjusted appropriately for 40 minutes and remove from suspension and dry the seedlings for ten minutes before planting in to pot.

5.   Transplant immersed seedling to the pot size of 20 cm diameter and 15cm  height with full of sun sterilized soil media which is composed with sandy soil, animal dung and forest or fertile soil in the ratio of 1:1:2 respectively.

6.   Incubate infected and untreated  seedlings at 26 + 2 °C and with 60%-80% Relative humidity for 48 hours for both treated and untreated seedlings.

7.   After removing the seedlings from the incubation, water was applied regularly to maintain the substrate at field capacity level and all treatments were at recommended condition except inoculated pathogen.

8.   Evaluation of treatments were started after ten day post inoculation with parameter of plant height, leaf number, infected leaf number, disease severity and incidence.

2.5 Disease rating

Disease development in the greenhouse was evaluated 4–9 weeks after inoculation using a modified version of the disease severity rating scale for Fusarium wilt of banana (Carlier et al.,2002). The rating scale ranged from 0 to 5, with plants showing no internal symptoms scoring  0 and plants showing 100% vascular discoloration scoring 5. In the glass house disease incidence was scored according to the presence or absence of external disease symptoms.  Healthy plants were given a value of 0, while diseased plants were scored as 1.

2.6 Temperature and relative humidity

To determine the effect of temperature  and relative humidity on fungal growth, mold incubator were used in laboratory and in glass house humidifier were used to adjust the normal environment for the pathogen.

Table 1: Average Temperature and Relative humidity during inoculation

3.  RESULTS AND DISCUSSION

3.1 Results

3.1.1   Effectiveness of Foc Inoculation Methods.

Untreated seedlings did not show wilting symptoms, while inoculated seedlings showed various degrees of wilting at 10 days after inoculation. The colonies of Foc were successfully re-isolated from inoculated poyo showing the symptoms of yellowing as seen in Figure 3(f- re cultured fusarium spores under microscope). Compared to other inoculation methods the optimized inoculation method under this experiment shows the highest disease intensity and score of the symptoms (Table 2). Ten seedlings of poyo variety  were evaluated for this experiment and typical symptom development of Fusarium wilt were observed successfully. The incubation period (the period from inoculation to the first symptoms appearance )varied depending on inoculation procedures, banana genotypes used, aggressiveness of the Foc isolate or environmental conditions.

3.1.2 Effects of Foc on disease incidence and severity

The Foc inoculation by dipping the plants at 1x 10⁶ conidia/ml were equally able to cause the wilting symptoms on ten tested poyo seedlings. At 10 days after inoculation, the infected seedlings showed  the score of the symptoms was lower  as shown in second score in (Table 2). Both at 3^rd and 4^th score of  DAI, inoculation with 10⁶ conidia/ml resulted an equal percentages of plants showing the symptoms (Table 2). However, the damages due to Foc infection has reached xylem tissues. Transversal sections of Foc un inoculated banana root in the control was presented in Figure 3A shows no symptom. Phenotypes of banana plants at 60 DAI showing wilting symptom score 5 or all inoculated seedling were dead  (Figure 4). This shows the success of inoculation protocol in Ethiopian condition.

3.1.3 Response of 10 poyo Cultivars to Foc Infection.

Un-inoculated poyo plants did not show wilting symptoms due to Foc infection. After inoculation of foc, percentages of banana seedling showing the symptoms were 100% (figure 4). All tested poyo cultivars showed higher average of score of wilting symptom and disease intensities than the control (Table 2). Based on disease intensities, ten poyo cultivars tested were identified as either very susceptible to foc. Plant height on pre and post inoculation revealed  high difference among seedlings as shown (fig, 5, 6 1a and 2d) respectively. On inoculated seedlings plant height decreases and plant number four on 3rd score already dead (fig 6, 2d). Plant height in un inoculated seedling increases about 10cm in period of experimentation (fig 6, 2d). 

Table 2: Mean disease severity and incidence

3.2 DISCUSSION

Inoculation method, age of plant, plant environment and amount of pathogen have  impact for the development of disease symptom. In this experiment disease severity increases during different scoring stages. under this experiment there is high variation among treatments and almost all inoculated banana seedlings show wilting symptom and lastly all seedling were died.  Un inoculated seedlings showed no wilting symptom and showed normal growth with deep green leaf.   The success of Foc infection in banana were the final results of a complex processes involving at least five stages, namely: (i) recognition by Foc of signals released by banana roots, (ii) Foc attachment on the banana root surface and penetration of Foc hyphae into root tissues, (iii) penetration of Foc hyphae into cortex tissues and degradation of physical defence system of root tissues, (iv) proliferation of Foc hyphae and production of microconidium in xylem tissues, and (v) secretion of fungal toxin and hydrolytic enzimes by Foc resulting in further tissue damages of the infected roots (Di Pietro et al. 2003). Wounding of root tissues assisted initial process of Foc infection in banana. Infection of Foc in banana might also through similar processes and wounding might  assisted Foc infection.

In the field, banana plant tissues might be wounded because of fallen  trees due to strong wind, pruning of suckers, damages due to sucking insects or nematode infection.(http://www.plantmanagementnetwork. org/pub/ php/management/ banana/ panama).            

Djatnika et al. (2003) stated that Foc is a soil-borne pathogen. It infects banana plants through lateral root hairs, and is able to infect root tissues through wounding due to infection of Radopholus similis nematode. Foc inoculation using optimized inoculation  method resulted more plants showing Foc infection symptoms and higher disease intensity. Wounding of banana root tissues followed by dipping of the wounded plants in the suspension of Foc conidia in optimized method guaranteed high Foc infection. Wounding has been shown to assist fungal penetration into inoculated plant tissues (Yusnita & Sudarsono 2004; Sakamoto & Gordon 2006). Observed wilting symptoms of banana seedling were the results of blocking of xylem tissues by Foc mycelia and by induction of thylosis because of activities of secreted enzymes and fusaric acid by F. oxysporum (Salerno et al. 2000). Infected root tissue into brownish or blackish colour could indicate the presence of damages in the root tissue of banana caused by Foc inoculation.  Although root damages at 10 DAI reached only the yellowing the lower part of the leaf, the tested poyo cultivar has shown average scores of wilting symptom ranged from 0 to 2 in different scoring time.  The functions of the cells were associated with water transportation and oxygen storage in roots. Cortex role did not directly related to water transportation. However, the cortex damaged might indirectly affect water transportation from root to shoot and leaf tissues and result in wilting symptoms. At 30 DAI, the percentage of banana plants showing wilting symptoms and disease intensities due to Foc infection were higher than that at 10 DAI. The increase of disease intensity at 30 DAI might probably be the result of damages in root xylem tissues due to Foc infection. The xylem was responsible for water and nutrient transportation in plants. The damaged xylem might have direct effects on water transport from roots to leaves; therefore, it might also have direct effects on causing wilting symptoms.  Under this research using developed optimized inoculation  methods, all tested poyo cultivars  were identified as very susceptible and un inoculated seedling shows no  Foc infection .

4. CONCLUSIONS AND RECOMMENDATIONS

Based on the data from this research, it can be concluded that inoculation method by wounding banana roots followed by dipping wounded seedling on suspension of 1 x10⁶ Foc conidia/ml  can be used to screen  banana germplasms against Foc.  Based on the value of observed disease intensities, ten poyo cultivar grown were identified as very susceptible to Foc infection.

This experiment confirms the protocol we followed to be efficient and reliable in terms of artificial inoculation of fusarium wilt of banana.  Optimized  protocol can be used for  screening of banana varieties that can resist/tolerate  fusarium wilt. Moreover, this protocol can also be tested for enset fusarium wilt.  This research recommend that at the end of inoculation process avoid visiting disease-free areas such as acclimatization rooms, or other banana experiments after working in Foc inoculation area and Once the experiment is finished, carry out extensive sterilization of pots, substrate and other tools used in the experiment. Take care of harvesting the pathogen and harvesting tools. Inoculated and un inoculated seedlings should be transplanted at different time. Start with untreated and proceed to treated one to remove contamination.

Introduction of banana materials should pass through quarantine system and Basic studies should have to be conducted on molecularly characterizing the races in the country. Varietal screening should be conducted to replace susceptible one and Country wide survey is important for early detection.

ACKNOWLEDGMENTS

The authors wish to acknowledge the Ethiopian Institute of Agricultural Research for the financial and logistic support during surveys and laboratory work.

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