The project inception meeting and workshop was held on 22 - 26 August 2006 at Bukit Tinggi, West Sumatra, Indonesia, hosted by ITFRI. Seventeen participants from ITFRI, Bioversity - Philippines, QDPI&F and ACIAR attended. During the meeting, the project's objectives were reviewed and its workplan developed.

Survey, Collection and Characterization. A training-workshop on soil health indicators and survey methodology was conducted at ITFRURI in Solok, West Sumatra on 6-10 November 2007. Forty-two staff of ITFRURI participated. The training was organized and facilitated by Tony Pattison of QDPI&F. Researchers were trained to operate the soil health indicator kit as well as to interpret the results. A training manual was produced and circulated.

Survey and collecting missions of Foc-infected plants were conducted from December 2006 to April 2007 by project staff of ITFRURI, Bioversity - Philippines and QDPI&F. The missions covered eight major banana producing regions/ provinces of Indonesia: Aceh, West Java, West, Central and South Kalimantan, North and Southeast Sulawesi and Papua. Eighty-five plant samples infected with Fusarium wilt (caused by the pathogen Fusarium oxysporum f. sp. cubense or Foc) were collected from the survey sites. Based on initial survey analyses, 16 host varieties were infected with Fusarium wilt across the surveyed locations. The most common varieties infected were 'Barangan' (AAA), 'Raja Siem' (ABB), 'Kepok' (BBB) and 'Ambon Putih' (AAA). Aside from Fusarium wilt, blood disease and Banana Bunchy Top Virus (BBTV) disease were also observed. The samples were brought to ITFRURI for processing and sending to QDPI&F.

An important output of the survey and collecting missions was the collection of 11 new banana accessions consisting of wild, diploid, triploid and tetraploid plants. These materials are now maintained at the genebank of ITFRURI, the designated National Repository and Multiplication Center of Indonesia and will, eventually be taxonomically characterized.

A total of 111 pure isolate-Foc samples from ITFRURI were sent to QDPI&F for VCG and DNA characterization. The first batch was composed of 60 purified isolates while the second consisted of 51. Twenty-four isolates of the first batch were analyzed for volatile production and VCG characterization. Most samples were positive for VCG 1213/16, the VCG of Foc Tropical Race 4 (TR4). DNA from 50 of the 60 samples in the first batch were extracted and quantified, while the rest were either contaminated or returned. The second batch of samples are being prepared for characterization. All samples are to be freeze-dried for long term conservation.

Virulence-host resistance study. Preliminary preparations have begun. Twenty-five accessions of diploid, triploid, and tetraploid wild and cultivated varieties were chosen for this study. The plant materials are currently being propagated in vitro at ITFRURI. The Fusarium wilt inoculation technique/protocol was validated and optimized at ITFRURI from October to December 2006 using the 'Barangan'/ 'Lakatan' variety. VCG 01216 was used in the preliminary inoculation test.

On-farm disease management demo-trials of disease management strategies. Project staff of Bioversity, QDPI&F and ITFRURI conducted participatory planning workshops with farmers and local extension agents in Kedondong, Lampung, Sumatra, and in Dampit, Malang, East Java on 27 May - 2 June 2007. About 25 farmers participated in each site. The workshops included an initial assessment of farmers' practices in banana disease management and production systems. Consequently, planting materials and disease management tactics (i.e biocontrol agents) for the demoplots are being readied by ITFRURI in preparation for the start of the field trials in October or November 2007. To complement this activity, the project coordinator met with the provincial officers of the Balai Pengkajian Teknologi Pertanian or BPTP (Assessment Institute for Agricultural Technology) in Lampung and Malang. An agreement was reached wherein BPTP extension staff would provide local technical support and supervision to the trials. Additionally, the project team visited a private banana commercial plantation (PT Nusantara Tropical Fruits, or NTF) in Lampung on 28 May 2007. It was discussed with NTF the posibility that they would participate in field trials to evaluate the project's proposed disease management strategies under commercial Cavendish plantation conditions where TR4 is a serious problem. This gives the project a broader scope involving both farmers' fields and a commercial plantation.

Progress summary
Our project objectives, regarding mitigating the threat of banana Fusarium wilt remain the same.
Survey, Collection and Characterization
Indonesia
The survey and collection of samples within the framework of the CP/2005/136 project is complete in eight major Indonesian banana producing provinces (See Section 2). One hundred and eleven Fusarium oxysporum f.sp. cubense (Foc) pure isolates were sent to QDPI&F for VCG (Vegetative Compatibility Group) and DNA characterization. The Foc isolates came from 16 different banana varieties.
To fully map the Foc distribution in Indonesia, provinces under the Indonesian / ACIAR bilateral project (CP 2004/034) are being surveyed to complement surveys under project CP/2005/136. ITFRI is generating pure isolates from the samples collected, to be sent to QDPI&F for VCG and DNA characterization.
VCG analyses at QDPI&F show that 73 out of the 111 isolates gave positive VCG results, belonging to several VCG groups (See Section 2). 44 out of the 73 VCG-positive isolates showed specific results for VCG 01213/16, the VCG that is associated with the virulent Tropical Race 4 (TR4). The remaining 38 isolates were negative in the VCG tests. There was a confirmation of VCG 0126 on a wild banana, Musa schizocarpa, collected from Papua Province. Nine new Musa accessions were collected from Central Kalimantan, North Sulawesi and Papua.
Papua New Guinea (PNG)
Survey and collection activities are being conducted in PNG. Six field surveys, yielding 36 samples were completed between September 2007 and February (See Section 2) These were sent to QDPI&F for testing. Fusarium wilt-like symptoms were uncommon in these surveys, indicating that Fusarium wilt disease is not yet well established in PNG. QDPI&F tests show that none of the 36 samples from PNG was infected with Fusarium wilt disease. Survey and collections are scheduled for June 2008 in Sundaun, which borders with Papua, where TR4 has been previously reported.
A huge diversity of local bananas was observed in Manus (See Section 2).
Taxonomists from the survey-team collected new banana accessions from Manus (10) and Western province (6). New germplasm accessions are important potential sources of resistance for specific banana diseases, including Foc TR4. The accessions were added to the existing germplasm collection of NARI in Laloki.
Development of rapid molecular diagnostic tool
A key project objective is to develop an accurate and rapid diagnostic molecular technique test for Fusarium wilt. One hundred and two Foc-positive isolates from Indonesia were studied by extracting their DNA, using a commercial DNA plant extraction kit BioSprint Tissue Lyser. Tests showed that 47 samples were found to be positive for TR4, 35 to be negative, and 20 samples gave inconclusive results. The accuracy of the test on the 102 samples was computed at 80.39%, which is relatively low, thus test procedures will yet have to be refined.
Virulence-Host Resistance Test
Four local banana varieties, Ambon Kuning, Barangan, Kepok , and Raja Sere, have been initially tested for resistance to the six following VCGs (See Section 2): 01213/16, 0123, 0120, 01218, 0126, and 012415. The project will be conducting virulence tests on a total of 25 selected wild and cultivated banana varieties to eight VCGs (See Section 2). Preliminary results showed Fusarium wilt symptoms such as vascular discolouration, chlorosis and wilting were observed on inoculated plants. Other tested local varieties showed common symptoms such as yellowing and wilting of leaves even three months after inoculation. Despite some observed corm infection. VCG01213/16 was noted to be most virulent among the tested VCGs. These results indicate differences in disease resistance/susceptibility of the various test varieties, and possibly differences in virulence of the different VCGs.
On-farm disease management demonstration trials
The farmer co-operators participatory planning workshop assessed farmers' practices in banana production and disease management, as well as options for management of Fusarium wilt disease. From this workshop, the following are the 'best-bet' options for Fusarium wilt management: The use of:
disease-free planting materials obtained from tissue culture (TC) propagation;
varieties tolerant or resistant to Fusarium wilt such as FHIA-17, FHIA-21, FHIA-25, GCTCV 119, Ketan-01, and one susceptible variety Ambon Warangan (Cavendish subgroup;
burning rice-hull on infected mats or spot eradication;
good agronomic practices;
a biological control using Pseudomonas fluorescens, adopted from the farmers' field-school program;
Demonstration plots were established in Lampung, Central Java and Malang, East Java in cooperation with farmer-co-operators.
The farmers were trained on the use of clean planting materials from banana tissue culture and the management of tissue culture seedlings for field planting. One important aspect of the project is to introduce a sustainable TC delivery system to small-scale farmers sourcing from commercial laboratories. Private tissue culture laboratories (22) producing banana meriplants were identified across Indonesia. A meriplant delivery system and a management protocol for village nurseries is being developed.

Survey, Collection and Characterization
Indonesia
Survey and collections of banana Fusarium wilt samples have been conducted (for both HORT 2004/034 and HORT 2005/136) in Indonesia in fifteen major banana producing provinces namely: Aceh, North Sumatra, West Sumatra, Lampung, West Java, Yogyakarta, East Java, West Kalimantan, Central Kalimantan, South Kalimantan, East Kalimantan, South Sulawesi, North Sulawesi, Southeast Sulawesi and Papua. A total of 259 samples were collected in both surveys. A total of 178 samples (111 samples collected through the HORT 2005/136 and 67 samples from HORT 2004/034) were sent to QDPI&F for VCG and DNA characterization. Relevant information was also identified from the results of socioeconomic surveys of banana farmers. A total of 583 farmer respondents were interviewed. It was found that banana wilts are the main causes of low banana productivity, and that farmers often do not distinguish the difference between Panama Wilt and Blood Disease. More than 90% of the banana farmers are men; the farmers were found to have an average of 7yrs education, with less than 10% of the respondents claiming formal training on banana production. For banana production and management, none of the farmers use tissue cultured planting materials or chemical treatments. Most of the planting materials used in the areas were from suckers of their own plants and also from neighbouring fields, increasing the chances of spread of corm and soil-borne diseases such as Foc and BBD. One other very relevant information derived from the survey is the observation that banana bunchy top disease (BBTD) is rapidly spreading in several provinces. This would potentially complicate the already disease-constrained banana production by small-scale farmers. BBTD spreads also through suckers from infected mother plants.
Papua New Guinea (PNG)
Surveys were carried out in Morobe Province/ Markham Valley, Kainantu, North Solomon Province/ Buka, East New Britain Province (ENB), Manus and the Western Province. The last of the planned surveys in PNG in Sandaun province which borders with Papua, where TR4 has been previously reported was scheduled for 19-23 May 2008. To date, all samples from PNG that were sent to QDPI proved to be negative to Fusarium oxysporum f. sp. cubense.
Other disease problems observed during the surveys included Sigatoka complex (including Black Cross, Cordana and Freckles) and Marasmiellus rot. The widespread occurrence of Marasmiellus rot was also confirmed in recent surveys conducted by officers of the Northern Australian Quarantine Strategy (NAQS) and the PNG National Agricultural Quarantine and Inspection Authority (NAQIA) in Western (June 2008) and Sandaun Province (2007). Thirteen new banana accessions were collected during the survey in Sandaun province and added to the existing germplasm collection of NARI in Laloki. Development of a rapid molecular diagnostic tool
DNA extraction
Three methods were investigated to extract DNA from Fusarium oxysporum f. sp. cubense samples. The methods investigated were phenol/chloroform extraction, Puregene kit (Qiagen) and a Biosprint kit. DNA was successfully extracted from all 111 samples received from Hort/2005/136 and from 45 of 67 samples received from Hort/2005/034. The quantity of DNA extracted ranged from 1 ng/L to ~ 350 ng/L. The Puregene Yeast DNA extraction kit is able to extract high molecular weight DNA from Foc that is suitable for PCR and AFLPs.
TR4 PCR
A TR4 PCR technique developed by Bentley et al (1999) was adapted in this study. For project HORT2005/136, a total of 111 samples were tested. 56 (50.4%) tested positive for TR4. 47 (42.3%) tested negative and the remaining 8 (7.3%) gave an inconclusive result. For project HORT2004/034 a total of 45 samples were tested. 19 (42.2 %) tested positive for TR4 and 26 (57.8%) tested negative. Overall for both projects, 75 out of156 (48%) tested positive for TR4, 73 (46.8%) tested negative and 8 (5.1%) were inconclusive.
AFLP
AFLPs were successfully performed and analysed for 75 isolates, with 4 different primer combinations. DNA was extracted from selected isolates using the Puregene Gentra yeast kit. After Sel PCR, a smear with distinct bands ranging between 700bp to 100 bp was visible for all samples processed. Often, different banding patterns could be observed for different samples processed with the same primer set. When the sel PCR product was run on the CEQ 8800, these bands were visible as distinct blue peaks with a maximum intensity of 200 000 units, whilst the size standard was visible as red peaks. A total of 427 polymorphic bands were identified across the four primers used, giving an average of 106 bands per primer set.
Virulence-Host Resistance Test
Indonesia
The virulence of the various VCGs was tested against various local varieties in the screenhouse and in the field at ITFRI. Both tests were conducted to understand the pathogenicity/virulence of various VCGs to different banana cultivars, an effort to develop a "set of differential cultivars" that may serve as a diagnostic tool and guiding framework for disease management, particularly cultivar deployment. Screenhouse studies involved spore inoculation of tissue culture seedlings planted in plastic pots. External and internal symptoms were assessed. The project has completed the first trial of screenhouse virulence study on 9 local banana cultivars against seven VCGs: 01213/16, 0123, 0120, 0124/5, 01218, 0126 and 01219 with three replications (10 plants/ replicate). Preliminary results showed that among the various VCGs, VCG 01213/16 (TR4) was observed to be generally the most virulent based on disease severity index (Table 3), and other virulence parameters such as incubation period. It is interesting to note however that there is an apparent cultivar by VCG interaction. The screenhouse virulence/resistance study is being validated using VCG1213/16 in a field trial at ITFRI. The experiment is conducted using 25 varieties, 12 plants per variety, arranged in randomized complete block design (RCBD), and replicated in two blocks. Disease assessment is in progress. Adequate data are expected to be collected and analyzed through the end of 2009.
On-farm disease management demonstration trials
The on-farm disease management trials focused on production system improvement, by adapting improved production and disease management practices that may improve banana yield and productivity. These were established in Dampit (East Java), and South Lampung. The elements of intervention included the use of disease-free planting materials in the form of tissue culture, resistant varieties, and cropping systems. Two introduced resistant varieties (FHIA 17 and FHIA 23) were compared to two popular local cultivars (Ambon Kuning and Ketan-01). These varieties were evaluated as monocrop compared to mix-cropping systems. In Dampit, some farmer cooperators interplanted the banana plants with nilam, coffee and cassava, the popular crops in this area. In Lampung, ground nut and ginger were used as intercrops. Farmer cooperators compared suckers versus tissue culture seedlings. The establishments of the demo trials were delayed because of the unexpected delay in the availability and delivery of tissue culture seedlings.
Preliminary data shows that FHIA 17 and FHIA 21 are showing less infection of Fusarium wilt compared to the local cultivars, although these varieties are showing some infection of Erwinia rots. Intercrops are initially showing less infection than monocrops. When taken cared of properly, tissue culture planting materials are showing better growth than sucker-derived plants, indicating that better yield could be achieved using healthy tissue culture seedlings. Preliminary observations point to the great potential of increasing banana yield by planting healthy seedlings and application of good agronomy such as proper population density, fertilization, weeding in either monocrop or in mixed planting. The overall performances of the various interventions are yet to be fully evaluated. In order to gather sufficient data, an extension of the project until the end of 2009 without additional fund was requested.