Huanglongbing management for Indonesia, Vietnam and Australia

The first annual planning and review meeting was held in Hanoi, in February 2003.. Experimental protocols were planned for most project activities after relevant presentations from participants. Orchards were also visited at Cao Phong in Hao Binh province. Protocols were circulated for comment in May 2003 but progress during the year was hindered by travel restrictions related to regional uncertainty.

In September/October 2003 Dr Andrew Beattie (the project leader) and Dr Paul Holford visited the Indonesian Botanic Gardens in Bogor and Gadjah Mada University in Yogyakarta. During this visit potential sites for field experiments were inspected and plans for constant temperature facilities required for other experiments were reviewed. Dr Paul De Barro (CSIRO) suggested modifications to the facilities when he visited Indonesia in December 2003 to plan experiments and visit field sites. Construction of the facilities has commenced and completion was scheduled for March 2004. In late 2003 a decision was made to appoint an entomologist (Dr Zamir Hossain) from January 2004, to undertake project activities in Indonesia, and to help coordinate other activities.

Dr Holford and Dr Beattie visited Vietnam from 5 to 10 October 2003 to review project protocols and continue planning of experiments. Dr Beattie also discussed these plans with project personnel during other non-project visits. Dr Mabberley helped to prepare a list of plants (citrus and citrus allies) to test as hosts for huanglongbing and the Asiatic citrus psyllid (Diaphorina citri), and to resolve uncertainty about relationships between these plants-an issue vitally important for the assessments.

During 2004, significant progress was made in overcoming setbacks stemming from the impact of international events in 2003. Field sites were established for a major objective in Vietnam in which management strategies (including use of white mineral oils and imidacloprid, a systemic insecticide) for control of huanglongbing and its vector, the Asiatic citrus psyllid Diaphorina citri, are being compared. Screenhouse studies on the effectiveness of imidacloprid were also undertaken in northern Vietnam. Progress was also made towards resolving crucially important issues related to the identity of true species of Citrus and Citrus relatives in Vietnam. These issues arose in 2003 when it became clear that published descriptions of these plants are in many instances of questionable accuracy and not supported by 'voucher' specimens in herbariums. These issues are important for management of the disease and conservation of unique plant material.

In Indonesia, controlled environment and screenhouse facilities required for research and propagation of plants were completed in July and became operational in September-October. Personnel required for each of the project objectives were chosen in April and they commenced work in July. Sites for major field experiments were selected at three altitudes(50, 650 and 1300 m above sea level). These sites will be used to assess management practices and the impact of climate on levels of infestations of the vector and the rate of spread of the disease. Initial controlled environment studies on the impact oil white mineral oil deposits on the feeding behaviour of the vector indicated that deposits of 0.25% to 2% sprays significantly reduce feeding. Confirmation of these results will boost our confidence in the use of oil sprays to reduce transmission of disease through control of the psyllid (based on ACIAR CS2/1993/005 in China, ACIAR CS2/1996/176 research in Sarawak (East Malaysia), and subsequent research in Sarawak) and reduced feeding by adult psyllids. This should apply particularly to situations where it will be possible to maintain populations of the psyllid at low or negligible levels, such as in large orchards or smaller orchards in areas/localities with common management strategies.

The annual project planning and review workshop was held from 22-26 November in Yogyakarta. It was attended by more than 10 participants from Indonesia, four from Vietnam, three from China, one from Japan and three from Australia. Highlights of the workshop were presentations by UGM personnel, a valuable review of research on the disease and the vector in China, and preliminary evaluation of a simple field microscope for detecting disease-damaged tissue in leaves. This technique should prove very practical for field use, and as an alternative to expensive molecular techniques that can only be used in laboratories. It was originally used in Indonesia 20-30 years ago and its usefulness appears to have been overlooked with recent focus by scientists on the use of molecular techniques. The disease leads to degeneration, and ultimately premature death, of cells (known as phloem) that transport sugars and other chemicals in plants. In Indonesia, the disease is known as 'citrus vein phloem degeneration' disease.

Dr Zamir Hossian was appointed as an entomologist at the University of Western Sydney (UWS) in early January 2004 to help manage project activities, particularly in Indonesia. He spent 16 weeks in Indonesia during the year, helping with the selection of personnel to work on project activities at Gadjah Mada University (UGM) and assisting senior Australian (Prof Beattie and A/Prof Holford at UWS, and Dr De Barro from CSIRO) and Indonesian (Prof Susampto, Dean, Faculty of Agriculture, UGM, and Drs Siti Subandiyah and Andi Trisyono) with planning and supervision.

During 2005, significant progress was made in overcoming setbacks stemming from the impact of international events in 2003 and ongoing impacts of similar events on travel. The project was reviewed from 6-12 November by Prof Myron Zalucki (University of Queensland), Dr Nguyen Cong Thuat (Ministry of Agriculture and Rural Development, Hanoi, Vietnam) and Dr Baharuddin (Hasanuddin University, Makassar, Indonesia).

In southern Viet Nam, studies at the project field site at Cai Be in Tien Giang in the Mekong Delta in which application of mineral oils, pesticides and other management strategies are being compared continued. Populations of the Asiatic citrus psyllid and the incidence of huanglongbing were low, much lower than anticipated, an outcome seemingly linked to the presence of guava trees interplanted between citrus trees at the site. The implications for control of the vector and the disease will have major repercussions for citrus production throughout Southeast Asia should ongoing sampling and future experiments confirm the observations.

In northern Vietnam, growth of trees at the project field site at Cao Phong, where a similar experiment to the one at Cai Be, but without guava interplants, is being conducted, was slower than in the tropical south. Populations of the psyllid were very low throughout the year (3 were recorded) and seemingly related to low incidence of psyllid in the local area due to increased use, and more thorough applications, of pesticides in recent years. Infestations of the defoliating gold dust beetle (Hypomeces squamosus) were of concern in all treatments, and raised the possibility that high incidence of the weevil could be due to the widespread absence of the once common predatory ant Oecophylla smaragdina (golden weaver ant) in northern Vietnam.

In northern Vietnam, limited surveys were undertaken to locate and collect material from endemic Citrus spp. and Citrus relatives. This work was done in conjunction with placement of an Australian Business Volunteer (Mr Harry Jones) at the National Institute for Plant Protection (NIPP). Prof David Mabberley (University of Washington) visited Hanoi shortly after Mr Jones returned to Australia. During this visit he examined plant material collected by Mr Jones, visited Cuc Phuong National Park 80 km to the west of Hanoi and the Science & Technology Research Institute, and met with Dr Dzuong Duc Huyen (Head of Botany and the local co-ordinator for PROSEA - a largely Dutch initiative linked to the University of Wageningen) with whom he discussed herbarium records in Vietnam.

Experiments on the impact of deposits of 0%, 0.25%, 0.5%, 1% and 2% of aqueous mineral oil emulsions of two mineral oils on oviposition by Diaphorina citri were also undertaken in screen-houses at NIPP. Deposits of emulsion with = 0.5% oil reduced oviposition on buds shorter than 6 mm and longer than 10 mm by more than 75%, with effects more evident for the nC24 agricultural mineral oil tested than for the nC21 horticultural mineral oil tested. Mortalities of eggs laid on deposits, and of first instar nymphs that hatched from eggs laid on the deposits, were also significant: total mortality for the two life-cycle stages in treatments with = 0.5% oil exceeded 55%.

In Indonesia, preparation and establishment of all field sites was completed in November. These sites are being used for research on: (a) the impact of altitude (40 m, 670 m and 1300 m asl) on the vector and the disease; (b) the susceptibility of Citrus and Citrus relatives to the disease (c) studies on natural enemies of the vector; and (d) the impact pest management strategies, including the use of mineral oils and insecticides. Experiments in screen-house and constant environment facilities at Universitas Gadjah Mada focused on feeding preferences psyllid adults on citrus relatives, and the impact of aqueous mineral oil deposits on feeding and oviposition by adults on mandarin plants in choice and non-choice assays. In the host-preference studies feeding on Swinglea glutinosa was significantly higher than on the citrus relatives tested. The next most preferred plants were Aegle marmelos and Murraya paniculata, then Limonia acidissima, Triphasia trifolia and Glycosmis pentaphylla. Males showed least interest in feeding on Murraya exotica, but no species was rejected for feeding.

In the choice tests with aqueous oil emulsions applied to mandarin plants, the proportion of adults landing on the plants treated with 0%, 0.25%, 0.5%, 1% and 2% oil emulsions was 49%, 18%, 17%, 9.4% and 6.4% respectively. Avoidance was more noticeable in the no-choice test. Mortality of adults 1 d after they were caged with plants sprayed with 0.25%, 0.5%, 1% and 2% oil emulsions was 22.5%, 37.5%, 40%, and 65% respectively: 100% adults died 2 d after they were caged with plants treated with 2% oil. Mortality appeared to be related to reduced feeding on plants sprayed with oil.

In southern Viet Nam, studies at the project field site at Cai Be in Tien Giang in the Mekong Delta in which application of mineral oils, pesticides and other management strategies are being compared continued. Guava interplants within the site continued to limit ingress of huanglongbing and its vector, the Asiatic citrus psyllid, into the site. The level of infection three years after citrus trees were planted reached about 20%. This compares well with about 24% two years after planting in French and Vietnamese studies at Cai Be in which the systemic insecticide imidacloprid was applied initially as soil drenches and then directly to the trunks of trees. The impact of guava on spread of the disease has the potential to radically alter the economics of citrus production in the Mekong Delta, and in similar environments, through additional income from sale of guava fruit, dramatic reductions in pesticide use, and, through increased longevity of citrus orchards, increased income from the sale of citrus fruit. The impact of guava interplants on spread of the disease has gained worldwide attention. In northern Vietnam, growth of trees at the project field site at Cao Phong, where a similar experiment to the one at Cai Be, but without guava interplants, is being conducted, continued to be slower than in the tropical south. No psyllids were observed within the site during 2006 and symptoms of the disease were not observed. Infestations of the defoliating gold dust beetle (Hypomeces squamosus) were, as in 2005, of concern in all treatments. Mineral oil sprays gave good control of citrus leafminer (Phyllocnistis citrella).

In Indonesia, plants in all field sites established in late 2004 grew slowly until the end of the extended dry season, and then quite rapidly. No psyllids were observed in these sites at mid (670 m asl) and high (1,300 m asl) altitudes. No psyllids were observed in mature orchards at 1,300 m asl, or in D-vac samples from these orchards. The major impediments to citrus production at this altitude appear to propagation on non-disease free plants, and inadequate, but relatively easy, control of heavy citrus powdery mildew (Oidium tingitaninum) infections and citrus leafminer (Phyllocnistis citrella) infestations. The diversity and abundance of predatory coccinellid species varied consistently with altitude. The incidence of the predators was highest at the lowest altitude and lowest at the highest altitude. Black citrus aphid (Toxoptera citricida) was the main host of the coccinellids, and heavy infestations were observed at all altitudes. Both primary parasitoids of the psyllid were recorded, with the endoparasitoid Diaphorencyrtus aligarhensis being more common than the ectoparasitoid Tamarixia radiata. The favoured hosts of the psyllid among 20 plant species and cultivars being compared at Purworejo (40 m asl) were, from October 2005 to July 2006, Citrus - junos, then Swinglea glutinosa and Murraya exotica. During the same interval nymphs were most common on Citrus hystrix, M. exotica, S. glutinosa and Bergera koenigii. More than 240 adults and 420 nymphs were recorded over the 9 months. At this point, M. paniculata, as a host of adults, ranks thirteenth among the 20 plant species and varieties in the trial, and no nymphs were recorded on any of the 16 M. paniculata plants in the trial. These results were unexpected, as M. paniculata is considered to be the favoured host of D. citri, and we are not certain if plants named as M. paniculata represent a single highly variable species, a hybrid (M. exotica), or two species (M. paniculata and M. exotica), as the validity of M. exotica as a species is uncertain. However, in our studies, significant differences in morphology and molecular biology were detected between the two species as named and M. exotica is growing and flowering more prolifically at Purworejo than M. paniculata. These findings are important as it now clear that M. exotica is susceptible to huanglongbing whereas M. paniculata is possibly not or is asymptomatic and plants grown as M. paniculata in China, Florida and Brazil resemble M. exotica.

No psyllids were recorded in the Purworejo where six treatments are being compared for control of the psyllid. However, some 20 sprays have been applied in the oil treatments since late 2004. No oil-induced phytotoxicity as been observed but some minor, not readily noticeable, oil soaking has occasionally been observed along the midveins of mature leaves. Trees in the imidacloprid treatment did not appear to be any better than trees in other treatments. Citrus greasy spot (Mycosphaerella citri) was less common on oil sprayed trees. Experiments in controlled environment facilities at Universitas Gadjah Mada demonstrated that psyllid females can detect mineral oil deposits on treated surfaces before landing, most probably though detection by antennal receptors of volatile oil molecules.

Studies on huanglongbing and its vector, the Asiatic citrus psyllid (Diaphorina citri) continued in Viet Nam and Indonesia. In southern Viet Nam, where applications of mineral oils, pesticides and other management strategies are being compared for control of the psyllid in an orchard at Cai Be in Tien Giang in the Mekong Delta, no differences have been detected between treatments. This outcome is due to the presence of guava trees inter-planted with king orange trees in the orchard. The level of disease in the orchard reached 24% in March 2008, increasing slightly from 20% in early 2007. This level of disease is dramatically lower than in orchards in the absence of guava. The psyllid has not been observed in the orchard, or in similar orchards, by University of Western Sydney personnel, scientists and citrus growers from Florida (April 2007), and scientists from China (May 2008), whereas, psyllids are easily found in orchards were guava trees are not planted. As a result, evaluation of guava interplants has commenced in northern Viet Nam, Indonesia, Florida and China; work in China has shown that guava volatiles repel adult psyllids. Other sources of volatiles are being considered and use of guava and other plants as groundcovers in inter-row spaces has been contemplated.

At Cao Phong in Hoa Binh near Ha Noi in northern Viet Nam, no psyllids were observed in the project orchard where applications of mineral oils, pesticides and other management strategies are being compared. The absence of the psyllid was most probably due to high pesticide use in adjacent orchards and to climate (Cao Phong is cooler than Cai Be, where the potential for psyllid populations to increase is higher).

In Indonesia, application of foliar fertilisers was shown to reduce disease severity in mandarin trees. Other studies have demonstrated that the psyllid populations and incidence of the disease are related to altitude: higher at low altitudes (10 to 60 m asl), uncommon at medium altitudes (600 m asl) and absent at high altitudes (1,300). Species abundance and diversity of natural enemies also varied with altitude. Studies on the influence of mineral oils on the behaviour D. citri continued to show that oil deposits reduce feeding and egg-laying by adult psyllids. Psyllid populations, though increasing, remained too low (due to slow ingress from surrounding orchards) during 2007 and early 2008 to determine differences between treatments in the field experiment where applications of mineral oils, pesticides and other management strategies are being compared.

Ongoing reviews of literature continued as part of the project and for preparation of an incursion management plan, for both the disease and its vectors, for the Australian citrus industry. These reviews indicate that Citrus evolved in Australasia, not Asia, as widely accepted, and that huanglongbing did not, as widely accepted, originate in China. Plans for extension activities from 2009 were considered at an annual workshop in Indonesia in January 2008. The workshop was attended by extension personnel and farmers, and inter-planting of guava in citrus orchards in Java, and more widely in Indonesia, commenced in preparation for these extension activities. Supplies of a mineral oil were also arranged for these activities.

Interest in the project led to the chief investigator (Dr Beattie) being invited to the annual Citrus Expo in Florida in August 2007, where he addressed some 700 people, mostly growers. Interest in the project and Australian expertise also led to Dr Beattie being invited by the United States National Science Academy to participate in a meeting in April 2008 to determine priorities for research on huanglongbing and D. citri in Florida.