Overview Objectives

The project involved Australian experts who demonstrated conventional and molecular technologies to diagnose a range of soilborne fungal diseases of important crops and determine possible controls, while working with local scientists to train and prepare them to continue the work.

Project Background and Objectives

Soilborne fungal pathogens cause serious crop losses in both tropical and temperate regions, with each climatic zone tending to favour a different suite of species. The fungi can build up in the soil slowly and insidiously over many years. Diagnosing soilborne pathogens, identifying them to species level, and testing for pathogenicity is generally much harder than for fungi causing leaf infections. Soilborne fungal infection may cause very general symptoms to the parts of the plant above ground - such as reduced yield, wilting or leaf fall - which may not be obvious to inexperienced observers as signs of infection.

The picture is further complicated by the fact that many of these observable symptoms may be caused by other plant stresses - mineral deficiencies or toxicities, climatic stress or nematodes - which may be operating at the same time. Moreover, such stresses may in themselves exacerbate the fungal infection or predispose to it. Environmental factors may also affect the pathogen. In northern Sulawesi, cloves, vanilla, sago and maize, all important domestic or export crops, suffer what appear to be soilborne fungal diseases, severely reducing their productivity.

Progress Reports (Year 1, 2, 3 etc)

The purpose of this project is two-fold: 1) to develop within the eastern region of Indonesia the capacity to diagnose diseases of agricultural crops, to study the epidemiology of these diseases, and to establish appropriate control strategies, 2) to establish training and research capacity pertaining to the above aspects, with a base at Sam Ratulangi University in northern Sulawesi.

Four main crops, cloves, vanilla, maize and sago, have been initially identified as the focus of this project. These crops were selected in consultation with counterpart scientists because they are domestic and export crops of economic significance and their productivity is severely affected by diseases thought to be caused by soilborne fungal pathogens.

The collaborating research institutions are The University of Sydney (commissioned organisation), Royal Botanic Gardens, Sydney, and Sam Ratulangi University, Manado, Sulawesi. There is extensive and experience with diseases of soilborne fungal pathogens at The University of Sydney and RBGS. This expertise has been developed through on-going research studies in Australia and Vietnam, including work in Australia on eucalypt, orchid, cereal and palm diseases of relevance to clove, vanilla, maize and sago problems respectively. Sam Ratulangi University is the major centre for training plant pathologists in north-eastern Indonesia. In addition, valuable support and advice is provided by Prof. Michael Wingfield, Forestry and Agricultural Biotechnology Institute (FABI), Pretoria, South Africa. Prof. Wingfield is a renowned tree pathologist with extensive expertise on vascular and foliar diseases of relevance to clove problems..

Outputs anticipated from this project include:

A comprehensive checklist of fungal diseases of the key crops and basic checklists of other less important crops.
An assessment of the economic importance of each of the diseases.
An understanding of the distribution, epidemiology and population dynamics of the diseases, which leads to the implementation of management strategies which minimises yield losses caused by these diseases.
The establishment of a basic diagnostics laboratory and a core of well-trained plant pathologists experienced in fungal diseases, their diagnosis and control.

During the first year of the project two trips to northern Sulawesi have been made by Australian scientists. Disease surveys, collection of samples, isolation of disease associated organisms, on-site training of local scientists have been conducted during these trips. A further understanding of the distribution, severity and causes of diseases affecting the main crops, in particular cloves and vanilla, has been achieved. More than 150 fungal isolates have been obtained from these crops and pathogenicity tests have been planned as subsequent studies. Collaboration with Prof. Wingfield has been established, working on the clove decline problem. An Indonesian scientist has visited Sydney for 2 months undergoing intensive training at the University of Sydney and Royal Botanic Gardens. A plant pathology laboratory has also been planned and designed with the space provided by Sam Ratulangi University. A full-time Research Fellow for the project has also been appointed, to commence duties in March 2002.

Immediate future work includes pathogenicity studies of clove decline and vanilla stem and root rot, further disease surveys and sampling for population studies, completion of laboratory set-up and establishment of greenhouse facilities, and arrangement of post-graduate enrolment for 2 Indonesian scientists. To facilitate these, 2 trips to Sulawesi will be made and several items of equipment will be delivered to Sam Ratulangi University and made operational.

The overall aim of this project is to develop research and plant disease diagnostic capacity in east Indonesia, in particular North Sulawesi. Project objectives are two-faceted: research and training.

Research

Research activities conducted during the reporting period included disease surveys, collection of disease management information, isolation of pathogens, and pathogenicity tests.

Extensive disease surveys focusing on clove yield decline and vanilla stem rot were conducted throughout North Sulawesi. Disease incidence and severity was recorded, and discussions with farmers conducted. Clove pathogenicity tests were conducted on a range of fungal organisms isolated from diseased tissue. Results revealed that Ceratocystis fimbriata is the causal agent of yield decline. This represents the first diagnosis of this disease worldwide. Pathogencity tests on vanilla showed that stem rot in this region is mainly caused by Fusarium oxysporium. Proper diagnosis of these diseases facilitates a methodological approach to disease management, which is the focus of the latter part of this project. Pathogenicity tests also confirmed that Ceratocysitis finbriata enters the clove tree via the trunk through wounds, in particular those caused by the stem-borer, Haxamitodera semivelutina. Fusarium oxysporium was shown to be a typical soilborne pathogen.

As a result of further disease surveys and sampling, large representative populations of the clove and vanilla pathogens have been obtained. These are crucial requirements for subsequent studies to better understand the population dynamics of these pathogens as well as the epidemiology of the respective diseases.

Other fruit and vegetable crops surveyed during the reporting year were asparagus, banana, cabbage, cassava, chilli, corn, garden peas, nutmeg, onion, papaya, potato, rambutan and tomato. More than 20 diseases were recorded, isolations from some of which were also made.

Collection of information on disease management continued during the reporting period. This will form the basis for subsequent experiments on disease management strategies.

Training and establishment of facilities

Training of counterpart scientists were conducted before and during each field surveys. In addition, the project Research Fellow was stationed in North Sulawesi for more than 3 months during the reporting year to conduct in-house training on a) pathogen isolation, maintenance, identification, and storage; b) design of pathogenicity experiments, plant inoculation techniques, and disease assessment in greenhouse; c) laboratory management; and d) planning of research activities. Such training comprised lectures, discussions, hands-on laboratory work and conducting experiments.

Arrangements have also been made for two counterpart scientists to undertake post-graduate studies. Enrolment will be in 2003.

Establishment of the plant pathology laboratory, with 4 structurally separate sections, in Sam Ratulangi University was completed during the reporting period. All laboratory equipment was either delivered from Australia or purchased in Indonesia. A greenhouse was also constructed to facilitate pathogenicity tests.

Year 3 (01/01/2003-31/12/2003)

Other than routine disease surveys of general crops in North Sulawesi, research activities during the reporting year were mainly focused on further understanding of the two most important diseases in this region, clove yield decline and vanilla stem and root rot. A good knowledge of the biology of these diseases is crucial for the implementation of disease management strategies, which will be the focus of the following year’s activities.
A quantitative survey of the disease incidence and severity of yield decline revealed that >90% of clove farms throughout North Sulawesi were infected at various levels of disease severity. Pathogenicity results of Ceratocystis on clove from the previous year were verified on mature trees (>15 years) in the field. Symptoms were observed to be clearer on mature trees. The fungus had been initially identified as Ceratocystis sp., but with the aid of molecular techniques was described as a new species, C. polychroma sp.nov. (the scientific publication of this is in preparation).
It was also established that the relationship between the insect borer Hexamitodera semivelutina and the pathogen was not an obligate and intimate association. Infection of Ceratocystis was shown to occur in the field independent of the insect borer. Spores of the fungal pathogen were shown to be carried by other animals (insects, nematodes and mites) inhabiting and foraging within the borer galleries. It was also observed that not all trees affected by the insect borer were infected by Ceratocystis. However, field observations indicated that the progress of the disease within the tree after the initial infection was greatly enhanced by insect borer activities.
For stem and root rot of vanilla, the pathogen Fusarium oxysporum f.sp. vanillae was shown to have the ability to remain within the host as an endophyte without showing any internal or external symptoms. Such endophytes could become pathogenic when the environment is favourable for disease development. This has significant implications for the management of this disease as all vanilla vines in this region are vegetatively propagated. The pathogen was also shown to have a very low level of genetic diversity throughout North Sulawesi.
A secondary disease on vanilla associated with stem and root rot is anthracnose caused by Colletotrichum gloeosporioides. Since the Glyricidia shade trees used to support the vanilla vines the disease on vanilla. However, isolation and identification of the pathogen showed that the anthracnose symptoms on Glyricidia leaves came from a different Colletotrichum species.
During the year Ir Frans Rondonuwu and Ir Berty Assa attended a 10-week course at the University of Sydney. The course included conducting a small research projects, with the focus on mycological and molecular techniques commonly used in plant pathology research. Ir Frans Rondonuwu has now enrolled in a Master’s program at UNSRAT, co-supervised by the project leaders and collaborators. As well, Ir Arthur Pinaria was awarded an ACIAR John Allwright Fellowship and will undertake a PhD research program at the University of Sydney.

(This summary is taken from the final report as the project has reached the formal end of the original period. It has been extended and a more current final report will be submitted at the end of the extension phase)

A) Research
The key crops identified in this project are cloves, vanilla and corn.

Cloves
The main disease on cloves is clove decline. On the basis of isolations and pathogenicity tests, the project established for the first time the fungal pathogen Ceratocystis polychroma as the causal agent of clove decline. This fungal pathogen was taxonomically described as a new species. It was revealed that >90% of the clove trees within 68 farms surveyed throughout North Sulawesi were infected, at varying severity levels of 43 - 72%. Surveys included various phenological stages of this perennial tree crop. The pathogen is associated with the clove trunk borer found in this region, Hexamitodera semivelutina. Although infection of this disease occurs mainly on wounds caused by the trunk borer, it was also shown to occur on mechanical wounds. It was observed that this disease can be potentially spread by all insects inhabiting or foraging within the trunk borer galleries, while the progress of the pathogen within each individual tree trunk is facilitated greatly by the extensive spread of the insect gallery within the trunk. The pathogen reproduces readily and abundantly both sexually and asexually within infected hosts. The population of Ceratocystis polychroma was shown to have a high level of genetic diversity. Strategies to reduce the extent of this disease include the control of the trunk borers, elimination of insects in general within borer galleries, farm sanitation and sealing of wounds especially those caused by trunk borers. The use of fungicides is not cost-effective and efficacy is not clear.

Vanilla
Vanilla stem and root rot was diagnosed to be caused by the fungal pathogen Fusarium oxysporum f.sp. vanillae. This was in accord with previous studies in other parts of Indonesia and throughout the Asian Pacific islands. The mean disease incidence of 36 farms (including various phenological stages of this perennial crop) throughout North Sulawesi was 46%, with severity ranging from 19% to 73%. The pathogen was shown to be a typical soilborne vascular fungal pathogen that enters the host via wounds above and below ground level. It was also shown to be capable of being an endophyte without producing any internal or external disease symptoms, strongly implying the importance of planting with pathogen-free material. Both varieties of vanilla (Madagascar and Common) were shown to be equally susceptible to the disease. Not unexpectedly, a large proportion of clones was revealed within the pathogen population of this asexual fungus. However, the overall genetic diversity was found to be relatively high. The population of this pathogen was observed to be panmictic throughout North Sulawesi. Most of the pathogen isolates studied (63%) belonged to the same vegetative compatibility group, which could explain the high levels of genetic diversity. Although the use of fungicides was shown to have an effect on reducing disease severity, it is not cost-efficient. Recommended disease control strategies include planting with pathogen-free planting material, farm sanitation (especially the removal of diseased tissues), use of systemic fungicide upon planting, ensuring crop health and vigour at all times, judicious pruning of shade trees during raining and flowering periods and use of clove leaves to suppress soil inoculum potential. A secondary disease caused by Colletotrichum gloeosporioides was also found to be present in North Sulawesi, causing very similar symptoms. This disease, however, is not as devastating as Fusarium stem and root rot.

Corn
Corn stalk and cob rot, although present in North Sulawesi, was not observed to be an important disease. Although the pathogen (Fusarium verticillioides) is commonly present within healthy hosts, symptoms of this disease are observed only when the crops are stressed. Population analysis of this fungus, as an endophyte, throughout North Sulalwesi revealed a relatively high level of diversity. Genetic comparison between the North Sulawesi population of this fungus and those of north Vietnam and New South Wales, Australia, showed that these populations were not genetically distinct from each other, indicating that this fungus is most probably introduced into the North Sulawesi region.

Other Crops
In the general disease surveys, a list of 29 crops (including the 3 key crops) with 62 diseases, together with a rough indication of their economic importance, were recorded throughout North Sulawesi. The list is attached (Attachment 1).

B) Training and Capacity building
A plant pathology laboratory with five specialised work areas was established for the purpose of facilitating disease diagnosis capacity and enhancing research and teaching capacity in the discipline of plant pathology in Sam Ratulangi University. This entailed structural renovation and installation of laboratory equipment, air-conditioners, and security and safety elements. Additionally, a greenhouse was also constructed.

Training of Indonesian project personnel took on various forms. Intensive courses of advanced plant pathology (9-12 weeks) were conducted for three members of the Indonesian team at the University of Sydney. One team member was awarded the John Allwright scholarship to undertake his PhD program in the University of Sydney. Another member was enrolled in his Master of Agricultural Science program at Sam Ratulangi University. Specific training sessions before and during disease surveys and research experiments were conducted by project scientist for all seven team members. A two-week workshop on the biology of soilborne plant pathogens was conducted for scientists from various Indonesian provinces. A 3-day workshop on soilborne plant diseases was conducted for extension officers of the provincial agricultural department (North Sulawesi BPTP). Five undergraduate 4th year students carried out small projects associated with the overall project activities.

Publication in scientific journal
M. van Wyk, J. Roux, I. Barnes, B.D. Wingfield, E.C.Y. Liew, B. Assa, B.A. Summerell, M.J. Wingfield. (2004). Ceratocystis polychroma sp. nov., a new species from Syzygium aromaticum in Sulawesi. Studies in Mycology 50: 273-282

Conference presentations
Ceratocystis fimbriata associated with Clove Decline in North Sulawesi. 8th International Congress of Plant Pathology, Christchurch, N.Z. Feb. 2002
Fusarium Stem Rot of Vanilla in North Sulawesi. 8th International Congress of Plant Pathology, Christchurch, N.Z. Feb. 2002
Ceratocystis polychroma, a potentially important pathogen of clove. 3rd Asia-Pacific International Mycological Conference on Biodiversity and Biotechnology, Kunming, China. Nov. 2002
Fusarium Stem Rot of Vanilla in North Sulawesi (in Indonesian). First Indonesian National Symposium on Fusarium. Purwokerto, Central Java, Indonesia, Aug. 2004
Distribution of Vanilla Stem Rot in Minahasa. First Indonesian National Symposium on Fusarium. Purwokerto, Central Java, Indonesia, Aug. 2004
North Sulawesi: the crops, the diseases and the sweltering heat. Australasian Plant Pathology Society NSW regional meeting, Orange, Australia, Nov. 2004
Ceratocystis polychroma, a potentially important pathogen of clove. Congress of the Southern African Society for Plant Pathology, Hartenbos, South Africa, Jan. 2005

Electronic publication
A CDROM on soilborne fungal diseases of plants in Indonesia was published and distributed among workshop participants, Sam Ratulangi University staff members and extension officers of North Sulawesi provincial agricultural department (BPTP).

Workshops
Biology of Plant Diseases Caused by Soilborne Fungal Pathogens, Sam Ratulangi University, Manado, Indonesia, Feb. 2004
2 weeks (+ 2 field days)
13 participants from various research institutions and provincial agricultural departments in various Indonesian provinces
Fungal Soilborne Plant Diseases, Sam Ratulangi University, Manado, Indonesia, Dec. 2004
3 days (+ 1 field day)
6 staff members of BPTP North Sulawesi & 2 lecturers from Khairun University, Ternate, North Maluku

Newspaper articles (Komentar is the main Indonesian newspaper in North Sulawesi with a wide readership throughout the province)
Kurang Dana Penelitian, Faperta UNSRAT Terima Bantuan Rp 2,4 miliar. Komentar, 10 October 2002
[Initiation of ACIAR project in collaboration between the University of Sydney and Sam Ratulangi University]
Berhasil Diagnosa Penyebab Penyakit Gugur Daun - Kerjasama Fakultas Pertanian Unsrat dan ACIAR Australia. Komentar, 13 September 2003
[Diagnosis of clove decline]
Faperta UNSRAT dan ACIAR Australia Temukan Penyakit Vanili. Komentar, 25 September 2003
[Research on and diagnosis of vanilla stem and root rot]
Peserta Diajar Mengidentifikasi Penyebab Penyakit - Lokakarya ‘Soilborne Plant Diseases in Indonesia’. Komentar, 11 February 2004
[Workshop on soilborne plant diseases]

Should be no more than 750 words. Please note that the progress summary will be made publicly available on ACIAR’s website and in other communication materials. Do not include any commercially- or institutionally- sensitive material in this summary (this information should be included in 4.7).

This project is an extension of a previous project (2001-2004) on soilborne diseases of crops in North Sulawesi, focusing on vanilla stem and root rot and clove decline.

1. Disease management strategies for vanilla stem and root rot
In order to conduct greenhouse experiments on various control strategies, the use of highly virulent isolates and a realistic and optimum amount of inoculum is essential, as it was found in the first phase of this project that natural soil inoculum levels in the field were very low. During this reporting period, fresh isolates of Fusarium oxysporum f.sp. vanillae were obtained from infected farms and purified, and their virulence was verified on vanilla vines in the greenhouse. Experiments were conducted on a wide range of inoculum concentrations and established that a concentration as low as 150g inoculum (mycelium grown on paddy grain) per kg soil was sufficient to induce disease symptoms on vanilla stems. Control experiments in the greenhouse are currently underway.

2. Disease epidemiology of clove decline (Dissemination of the pathogen in the soil)
The first of two experiments has been conducted to investigate whether the pathogen of clove decline, Ceratocystis polychroma, is soilborne. Based on isolation from soil samples collected from plantations affected by clove decline throughout North Sulawesi, the pathogen was not found to be soilborne. This is in accordance with reports on various species of this genus in that dissemination of infective spores via soil is rare. This first experiment was conducted during the dry season. This will be repeated in the coming wet season.

3. Disease epidemiology of clove decline (Insects associated with cloves infected with clove decline)
An inventory of insects associated with diseased clove plantations throughout North Sulawesi was collated during the dry season based on insect collection from malaise traps and borer galleries on tree trunks. The inventory included insect species from 7 orders and 38 families. Not all insects were described down to the genus due to the presence of only larval stages. This inventory will be extended during the next wet season.

This project is an extension of a previous project (2001-2004) on soilborne diseases of crops in North Sulawesi, focusing on vanilla stem and root rot and clove decline.

Disease management strategies for vanilla stem and root rot.
All greenhouse experiments on control measures of vanilla stem and root rot have been completed during the reporting year. Both cinnamon powder and shredded clove leaves as soil amendment showed some levels of disease control. There was also evidence of disease resistance in four clonal lines obtained from the Research Institute for Spice and Medicinal Crops. No disease control was demonstrated using two commercially available fungicides, Thiaram and Mancozeb. These preliminary results conducted in the greenhouse will be transferred into the field as field trials, which are currently being set up.

Epidemiiology of clove decline (insect association)
An inventory of insects associated with diseased clove plantations in the wet season throughout North Sulawesi had been established earlier in the project. In this reporting period, this insect inventory list was extended to include insects collected in the dry season, which consisted of 45 families in 7 orders.

Disease epidemiology of clove decline (Insect association).
Following on from previous activities on establishing an inventory of insects associated with clove decline in addition to the clove borer, Hexamitodera semivelutina, a greenhouse experiment has been set up to determine whether Nitidulids beetles were able to spread infective propagules of the fungal pathogen, Ceratocystis polychrome, and result in disease infection. The experiment is on-going.

Disease management of clove decline (field trials).
Control experiments using insecticides, Carbofuran, Dimetoat, injected into clove tree trunks did not show any control of clove borer activities. Further experiments using Emamectin and Thiomectosome are underway. However, solid naphthalene applied as a slow-release fumigant showed good control of the clove borer.

Project Outcomes

Disease management strategies for vanilla stem and root rot In light of earlier evidence that Fov inoculum potential was very low in natural farm soil, an initial experiment determined the appropriate concentration of inoculum for use in control experiments. Subsequent greenhouse experiments established that the incorporation of cinnamon powder or shredded clove leaves into the soil effectively lowered Fov inoculum in the soil, thus reducing disease incidence. Clove leaves contain eugenol while cinnamon powder contains cinnamaldehyde, both of which have antimicrobial properties. Clove leaves, however, are more economical and more readily available throughout North Sulawesi. Experiments also showed that fungicides Thiaram and Mancozeb did not effectively reduce disease incidence. Two of four vanilla clones obtained from the Indonesian Institute for Spice and Medicinal Crops, Bogor, exhibited promising levels of resistance. Management strategies involving farm sanitation (removal of all infected plant tissue, removal of weeds and planting of disease-free material), soil amendment (incorporation of shredded clove leaves) and fertiliser application (chicken and cow manure) were established in farm trials that eventually became demonstration farms for vanilla growers.

Epidemiology of clove decline Experimental results revealed that the clove decline pathogen, C. polychroma, was not soilborne but was occasionally detected in soil due to the presence of infected host debris or spore-carrying insects. The clove trunk borer H. semivelutina was associated with the fungal pathogen by providing a favourable micro-environment in borer galleries for the proliferation of the pathogen. Masses of C. polychroma spores produced on fruiting structures were observed within borer galleries. The association between the fungal pathogen and the trunk borer was not found to be an intimate one. While borer larvae were not shown to be disease vectors, other invertebrates inhabiting the borer galleries (nematodes, mites as well as a myriad of other insects) were observed to carry spores of the pathogen. Insects trapped outside the trunk galleries on clove trees, in particular Nitudulids, were shown to carry propagules of the pathogen which were infective on cloves. A comprehensive inventory of insects associated with clove farms and trunk borer galleries was established over two seasons (rainy and dry), gathered from insect nets, traps and sweeping Malaise traps, and also hand-picked from borer gallery samples. The wet season inventory comprised seven orders and 38 families, while that of the dry season comprised seven orders and 45 families.

Disease management strategies of clove decline A series of control experiments tested the efficacy of injecting insecticides into clove tree trunks to eliminate trunk borers and other invertebrates associated with borer galleries. Liquid injections of Carbosulfan, Dimethoate, Emamectin and Thiamethoxam were all ineffective. Solid naphthalene, however, as a slow-release volatile insect fumigant used as plugs for borer gallery holes along tree trunks was highly effective in controlling trunk borers and other associated invertebrates. The naphthalene plugs also served as physical barriers preventing invertebrate passage in or out of the gallery holes, thus keeping the spread of the fungal pathogen in check. In addition to its effectiveness, naphthalene has the advantage over other insecticides in terms of lower cost, easy availability, low toxicity and ease of application. A demonstration farm using these naphthalene plugs was set up and monitored for insect control. Management strategies also include the removal and burning of severely infected or dead branches to control disease spread.

Farmer extension Three brochures on vanilla and clove diseases and disease management information were produced and distributed to farmers. Four farmer extension workshops (two on vanilla diseases and two on clove decline) took place in August 2007 at Sam Ratulangi University. Fifty-four vanilla farmers from 16 villages attended the workshops. Ongoing discussion and extension now takes place between farmers and staff from the AIAT, North Sulawesi and Sam Ratulangi University.

Project ID
CP/2000/094
Project Country
Inactive project countries
Commissioned Organisation
University of Sydney, Australia
Project Leader
Professor Lester Burgess
Email
l.burgess@agec.usyd.edu.au
Phone
02 93512526
Fax
02 93516481
Collaborating Institutions
Sam Ratulangi University, Indonesia
Royal Botanic Gardens, Australia
Assessment Institute for Agricultural Technology, Indonesia
Project Budget
$553,358.00
Start Date
01/01/2001
Finish Date
31/12/2003
Extension Start Date
01/01/2005
Extension Finish Date
30/06/2007
ACIAR Research Program Manager
Dr T K Lim