Diagnosis and management of wilt diseases of banana in Indonesia

PROGRESS SUMMARY
Funds were not available until June 2005.
Objective 1. Improved understanding of the epidemiology and biology of banana wilt diseases. Rapid progress was made in implementing PCR diagnostic technologies at UGM for Blood Disease Bacterium (BDB) and Fusarium oxysporum f sp cubense Tropical Race 4 (Foc TR4). In addition two IFRURI staff were trained for 4 weeks in Australia in PCR methodologies and mycological methods. Information on the distribution of BDB and Foc was collated by BPOPT and distribution maps were produced showing changes in disease occurrence from 2003 to 2005. Preparations for the field trials program on biocontrols and on varieties are in hand and propagation of plants is in progress. The targeted surveys were delayed due to administrative changes in the second half of 2005. These are scheduled to be implemented now.

Objective 2. Evaluate management options for wilt diseases
Collection and evaluation of data collected by regional extension bodies (BPTPH) on efficacy of their biocontrol preparations is in progress. Varieties with known tolerance or resistance to Foc TR4 and/or BDB have been nominated for propagation for field studies.

Objective 3. To extend best practice management practices for banana wilt diseases to growers
First pass recommendations for management of banana wilt diseases have been produced by the team and communicated to the Indonesian Banana Wilt Task Force. Good relationships have been forged with 7 regional BPTPH

1. Review of existing Indonesian knowledge on disease distribution and associated factors.
Completed in 2005.
2. Targeted surveys of the occurrence and severity of banana wilts and associated factors.
The targeted surveys are yet to be done and are scheduled for completion by ITFRI in 2007. The nine provinces to be surveyed are as follows: S Sulawesi, E Java, W Java, Lampung, W. Sumatra, N Sumatra, E Kalimantan, Jogjakarta, Aceh. The intention is to choose both diseased and apparently healthy sites in a range of situations and altitudes, with 5 sample locations in each of 3 shires in 3 districts per province.
3. Transfer of Foc PCR diagnostic and VCG characterisation techniques and BDB PCR diagnostics (recently developed in Australia) to Indonesia. Optimisation of BDB PCR methodology for plants and soil.
Following training of ITFRI and UGM staff in 2005, Dr Fegan made follow-up visits to UGM and ITFRI in February and June 2006 to supervise implementation of the PCR tests. The PCR tests were improved in several ways to increase reliability. The PCR test for BDB was also improved to detect BDB in dried vascular tissue taken from a diseased plant. This improvement means that this test can be carried out on the same vascular strands taken for the isolation of Fusarium oxysporum f. sp. cubense (Foc). Hence, there is no need to take multiple sample types to detect the BDB and Foc during the targeted survey.
4. Laboratory, greenhouse and field studies on epidemiology of wilt diseases caused by Foc and BDB and factors conducive to disease, including determination of the modes of infection of bananas by BDB, alternative hosts and survival time in soil, utilising conventional microbiological methods and molecular diagnostic tools.
Studies at UGM found that 4 species of Heliconia are hosts of BDB, while several solanaceous species that were tested were not hosts. The development of BDB disease at different altitudes with different humidity and temperature ranges after inoculation of soil with and without root wounding are also being assessed at UGM, with preliminary results suggesting that the higher temperatures at low altitudes favour faster disease development.
5. Characterisation and scientific naming of BDB
The genetic and phenotypic diversity of 30 strains of the BDB was assessed at UGM as a prelude to the formal naming of the bacterium. The formal naming work will be followed through at UGM using a strain that is typical of the ones studied..
Objective 2. Evaluate management options for wilt diseases
Alternative hosts
At UGM the ability of BDB strains to infect solanaceous hosts is being tested on tomato, eggplant and chilli etc. The isolates thus far tested are not able to induce disease on any host tested. These tests are ongoing.

At UGM 4 species of Heliconia spp were inoculated with BDB using infiltration of 1 ml BDB cell suspension at a density of 108 cfu/ml at the basal stem of Heliconia seedlings to study the pathogenicity of the bacterium on Heliconia plants. Wilting symptom development was observed for 6 weeks and at the last observation the basal stem was cut and open to observed any necrotized symptom due to the bacterial inoculation. Infection was confirmed by PCR techniques using specific primer of BDB. The results suggested that wilting symptom score ranged from 0 (no symptom) to 2 (with 2 leaves showing wilting, chlorosis, or necrosis) on H. bihae and there were 3 seedlings from 5 plant replicates showing wilting symptoms. The other species of H. collinsione, H. juguinii and H. hirsute, each with only one inoculated seedling showed mild wilting. The sections of basal stem showed that there was some necrosis symptoms in the vascular bundles. PCR tests confirmed the presence of BDB in the tissues.
Lab / glasshouse & field studies on survival of BDB in soil
The survival of BDB in different soil types was studied at UGM on Regosol (light soil structure), Andosol (medium soil structure) and Vertisol (heavy soil structure) inoculated with BDB cell suspension at 108 CFU/ml after incubation for 0 - 6 months then planted with susceptible plantain cv. Kepok. The results suggested that BDB could survive in the soil up to 6 months in the soil except in Andosol soil with drastically declining diseases intensity with period of incubation. The wilting intensity at 6 months after incubation ranged from 1.67% in Regosol, 0% in Andosol, and 6.67% in Vertisol soil respectively.
The effect of infected plant debris in the soil for BDB survival was studied at UGM by burying 200 g infected materials in 1 litre soil and incubating for 0 - 6 months before the mixture was planted with susceptible plantain cv. Kepok. After 5 and 6 months of incubation, the wilting intensity on the soil infested using infected fruit pieces were 21.67 and 15.00%, pseudostem pieces were 8.33 and 3.33%, pseudostem bulk on soil surface 6.67 and 0.00%, and BDB pure culture 5.00 and 0.00% respectively.
Studies to verify factors conducive to disease
The development of BDB disease at different altitudes with different humidity and temperature ranges after inoculation of soil with and without root wounding are being assessed at UGM. At low altitude (100m asl) the daily temperature ranged from 22-34C, at medium altitude (1000m asl) the daily temperature ranged from 20-30C, and at high altitude (1500m asl) daily temperature ranged from 10-25C. The relative humidity at low altitudes ranged from 52-91%, those at medium altitude ranged from 60-95% and those at high altitude ranged from 65-98%. The treatment of wounded seedlings resulted in higher disease intensity compared to unwounded seedlings. The disease intensity 3 weeks after inoculation on wounded seedlings at low, medium and high altitudes were 100%, 100%, and 88% respectively, whereas the unwounded seedlings resulted the disease intensities at low, medium, and high altitudes of 53%, 40%, and 12% respectively. These preliminary data indicate that BDB disease progresses more quickly at higher temperatures and humidity.
Verification of the composition, efficacy, potency, shelf life, application rates, and optimum timing of application of the biocontrol agents (particularly Trichoderma) that are currently in use in the field.
Following scrutiny by Dr Nasir (ITFRI) of locally-generated efficacy data, documentation and production systems on a range of Trichoderma formulations in several provinces, it was decided to use a formulation from Hasarudin University in South Sulawesi in each of the field trials as the "lead formulation", comparing its performance with a local formulation at each site.
Evaluation of efficacy of soil and plant treatments.
This greenhouse work was scheduled to begin at ITFRI in 2nd half of 2006. However little research was done due to administrative problems. Since the resumption of research activity in January 2007, there was a period of disruption of activity at ITFRI due to the Solok earthquake. Selected areas of this research will be carried out in 2007 and 2008.
Optimisation of promising treatments and field evaluation of existing lines of resistant/tolerant bananas.
Propagation of plants of selected lines for biocontrol field studies began in the 2nd half of 2006, tissue cultured plants were acclimatised at ITFRI ready for planting out in Q1 2007. Four trials are planned, two in West Sumatra at two altitudes, one in central Java and one in South Sulawesi. In each trial, the efficacy of a formulation of Trichoderma from South Sulawesi will be compared with a local formulation, on four banana varieties Barangan, Kepok, Puju and Tanduk.
Plants for the variety trials are currently being propagated (ITFRI)
Evaluation of efficacy of soil and plant treatments.
This work was scheduled to begin in greenhouses at ITFRI in 2nd half of 2006. However only limited research was done due to administrative problems. Selected areas of this research will be carried out in 2007 and 2008.
Optimisation of promising treatments and field evaluation of existing lines of resistant/tolerant bananas.
Propagation of plants of selected lines for biocontrol field studies began 2nd half of 2006, and plants were acclimatised at ITFRI ready for planting out in Q1 2007. Four trials were planned, two in West Sumatra at two altitudes (ITFRI), one in central Java (UGM) and one in South Sulawesi (ITFRI, with assistance from BPTPH and Prof Baharrudin from Hasanudin University. Three of the trials were established after the reporting period in January-Feb 2007 and the South Sulawesi trial was planted in March 2007. In each trial, the efficacy of the formulation of Trichoderma from South Sulawesi will be compared with a local formulation, on four banana varieties Barangan, Kepok, Puju and Tanduk.
Plants for the variety trials are currently being propagated. These trials will also be used as demonstrations of BDB control methods.
In addition, with the cooperation of the various BPTPH, plots will be established with existing resistant and tolerant varieties.
Objective 3. To extend best practice management practices for banana wilt diseases to growers
Liaison with the extension organisations in 9 provinces in Indonesia has continued. It is planned to utilise the Foc field trials as focal points for farmer discussion groups on both BDB and Fusarium wilts and other BPTPH extension activities.

Characterisation of strains and variability of BDB is nearing completion. Considerable progress was made in further improving the detection methods for Blood Disease Bacterium (BDB). A PCR method for detecting BDB in dried vascular strand samples was developed. It will enable detection of the bacterium in samples collected originally for the detection of Fusarium oxysporum f,sp, cubense (Foc). Studies on the host range of the bacterium showed that some species of Heliconia supported survival of BDB. Inoculation experiments showed that BDB, in addition to the well-known mode of infection via male flowers, can infect bananas from soil. Research on the survival of BDB in soil and plant debris showed that it declined to very low levels after 3 months and was not detectable after 6 months. These results have allowed the development of strategies to rehabilitate areas affected by BDB, including destruction and burial of infected plants, rotation to non-host crops, then re-planting with bananas.
Three field trials on biological control of Foc were established, with 4 banana varieties that had been propagated by tissue culture: Puju, Kepok, Tanduk and Barangan. Two of the trials were in highland and lowland areas of West Sumatra, while the third was established on land owned by the University of Gadja Mada in Jogjakarta. The West Sumatra trials were on farms with a history of wilt disease, while the trial at UGM was artificially inoculated with Foc. Data on disease incidence and the populations of Foc and the biological control agent Trichoderma were collected. Populations of Trichoderma after the biocontrol agent was applied rose initially, then declined to very low levels after three months, indicating that repeated applications may be required for efficacy.
When the banana plants flowered it became apparent that some plants in the trials were not of the designated variety. In particular the "Tanduk" plants at UGM were found to be cv Panjang, while in other plots one or several plants of one variety were in the same plot as another. The trials were mapped and individual plant data are being kept to enable separation of varietal effects.
Changes were made to pathways for funds transfer and to personnel, including the appointment of Dr Catur Hermanto as the principal researcher in Indonesia. A meeting of all participants in the project was held in July 2007 to review objectives, activities and progress.
The targeted survey of wilt diseases finally got underway and is in progress. A database has been developed to facilitate reporting and analysis of the data. Provinces surveyed to date are West Sumatra, Yogyakarta, East Kalimantan and East Java. A further five provinces are planned to be surveyed, and access to samples collected during the surveys of Foc in other provinces, carried out by the ACIAR multilateral project CP/2005/136, is being sought so data on the distribution of BDB can be added to the Foc results. Plants have been propagated for use in varietal trials in several provinces.