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IRCHLB Proceedings Dec. 2008: www.plantmanagementnetwork.org

A4 Huanglongbing (HLB) a graft transmissible psyllid-borne citrus disease: Diagnosis and strategies for control in Reunion Island - A case study (Translation from French of excerpts from the original 1988 Ph.D. Thesis of Benard Aubert, entitled: Le greening une maladie infectieuse des agrumes d’origine bactérienne transmise par des homoptères psyllidés Stratégies de lutte développées à l’île de la Réunion, Circonstances épidémiologiques en Afrique Asie et modalités d’intervention).

Huanglongbing (HLB) a graft-transmissible

psyllid-borne citrus disease

Diagnosis and strategies for control

in Reunion Island

A Case Study

Excerpts Translated from

Le greening une maladie infectieuse des agrumes d’origine bactérienne transmise par des homoptères psyllidés Stratégies de lutte développées à l’île de la Réunion,

Circonstances épidémiologiques en Afrique Asie et modalités d’intervention (1988)

Bernard Aubert

T. and K. Gottwald are warmly acknowledged for the final editing and corrections of the document. Many thanks also to P. Stansly for his help in reviewing the translation.

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Foreword Citrus huanglongbing (HLB) was first reported in India in the early 1900’s and was considered for some time by local entomologists as a debilitating problem linked to outbreaks of the Asian citrus psyllid Diaphorina citri Kuwayama. Further research conducted at the Lingnan Agricultural University of Guangzhou, China between 1941 and 1956, ascribed HLB to a psyllid-borne and graft-transmissible disease. The common name Huanglongbing (or yellow shoot disease) was given to this disease by local farmers of the Guangdong Han River delta. Independently an African form of HLB, also graft-transmissible and vectored by the African citrus psyllid Trioza erytreae (Del Guercio), was reported from South Africa in 1965 and was referred to as ‘Greening’ of citrus. By 1970, French plant pathologists of INRA1, maintaining a greenhouse citrus collection at Versailles with the support of the overseas institute IFAC2, found that the above Asian and African debilitating problems were associated with the presence of a bacterial organism that infested the phloem sieve tubes of affected plants. This was the first description of a phloem restricted bacterial organism in plants using transmission electron microscopy. In1994, the INRA-IBVM team isolated the 16S ribosomal DNAs of the African and Asian HLB strains, and placed these HLB bacteria in the alpha subdivision of the class Proteobacteria. Therefore, the interim taxonomy status for HLB became Candidatus Liberibacter africanus, Ca L. asiaticus, and a recent Ca L. americanus. None of these organisms have yet been isolated in pure culture. The HLB bacterium is believed to affect the cytoplasmic continuity between adjacent sieve tube elements, especially the functionality of sieve pores. Lower rates of translocation of carbohydrates and related metabolites triggers sequential traumas beginning with the yellow shoot syndromes of citrus leaves. Reduced source-sink-transfer of phloem sap leads to starch accumulation, leathery consistency of foliage and mineral-like deficiencies of the leaf lamina. Later consequences of infection are the disorganized proliferation of phloem in the leaf veins, fewer and shorter new flushes of foliage, premature leaf drop, atrophy of sexual organs with an off flavor of fruits. aborted seeds, root starvation leading to reduced feeder root system, reduced water conductance and finally gradual twig mortality followed by shoot dieback. The Asian form of citrus HLB abruptly reached the American Continent in the early 2000’s, causing immediate economic damage. The evolving pandemic advanced to the point of compromising the position of citrus as the most abundant fruit commodity ahead of grapes and banana, and the disease has taken the Western commercial citrus industries by surprise. Experts are actively reconsidering the various segments of their industry, including genetic selection, nursery production, orchard management and sanitation, integrated control, fresh fruit marketing, and even juice processing. Although previously restricted to inter-tropical Asia and Africa south of the Sahara, HLB outbreaks were surprisingly reported from the best and more intensively managed citrus areas of Brazil, Florida, the Caribbean and more recently from Mexico and California. The Mediterranean Basin is also considered threatened. The sudden spread of the Asian vector in combination with its Asian HLB strain is the result of man’s global activities, possibly combined with climatic changes, thus allowing vector and pathogen to move long distances. The results presented in this thesis translation are part of a research program conducted between 1973 and 1985 at IRFA3 Reunion Island, with the scientific support of INRA Bordeaux and of Bordeaux II University. Technical backing was also received from the SRA4 of Corsica. Most of this information remained unpublished in official scientific journals, although 100 copies of the original PhD thesis document were printed in 1988 by IRFA-CIRAD5 for the immediate beneficiaries of this research program. The sudden spread of the disease in the American continent, and the contacts established with the organizers of the International Research Congress HLB held in Orlando, Florida, in December 2008, prompted this English language presentation. In 2009, CIRAD was granted funding from the Florida Citrus Production Research Advisory Council (FCPRAC). The project aims to fulfil the last Koch’s of postulates, i) isolate the HLB bacterium in pure culture, ii) re-inoculate and obtain HLB symptoms, iii) re-isolate and obtain the same causal agent, so as to open new avenues of understanding/control of HLB pathogen-plant-vector relationships. B. A.

1 INRA (Institut National de la Recherche Agronomique) Laboratoire de Biochimie Centre de Versailles. This research unit was

transferred in 1971 to the INRA Centre of La Grande Ferrade (Bordeaux) and became the Laboratoire de Biologie Cellulaire et Moléculaire (LBCM) operating presently under the entity of Institut de Biologie Végétale Moléculaire.

2 IFAC (Overseas Research Institute for Tropical Fruits) created in 1942 with major activities on bananas and citrus in the Caribbean, America, Africa, Reunion and the Mediterranean Basin. In 1972 the name of this Institute became IRFA.

3 IRFA (Institut de Recherches sur les Fruits et Agrumes). In January 1985, IRFA was merged with the new French CIRAD. 4 SRA Station de Recherches Agronomiques de San Giuliano Corse, created by IFAC in 1958 and presently managed by a joint

team INRA-CIRAD. This Center hosts the French Certification Scheme for Citrus. 5 CIRAD (Centre de Coopération Internationale en Recherche Agronomique pour le Développement) Montpellier. The Research

Unit for the coconut heartrot and citrus HLB (CHCHLB) deals with tropical phloem-restricted plant pathogenic organisms.

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Part One Occurrence of HLB in Reunion, Symptomology and

Anatomical/Physiological Disorders 1. Botanical and Horticultural aspects of HLB in Reunion Rutaceous plants of Reunion and citrus germplasm tested for HLB

Reunion belongs to the archipelago of Mascareignas and is located near the Tropic of Capricorn at 21°5 South latitude and 55°29 East longitude. This two-million-year-old volcanic island was found to host nine indigenous species of Rutaceae represented by four genera: Xanthoxylum, Evodia, Vepris and Toddalia (Cadet 1977). Subsequent introductions by man over the last centuries included 10 additional genera of the subfamily Aurantoideae: Casimiroa, Clausena, Murraya, Mi- crocitrus, Triphasia, Aegle, Feronia,Fortunella, Poncirus § relatives of the genus Citrus. Citrus seeds

were imported by explorers, along with nutmeg and clove trees, in the mid 1770’s from Ambon Island (Moluccas), via Mauritius. Fusee Aublet (1775) produced the first catalog of these imported citrus relatives. He wrote official documents mentioning limes, pummelos, kaffir limes, sour and sweet oranges, as described by Rumphius (1750). Another early introduction of citrus into Reunion was the Malagasy Voangassaye, a mandarin of possible Indonesian origin, that escaped into the coastal rainforest of Reunion becoming feral, and whose date of introduction is unknown. The evolutionary isolation of the ecosystems in Reunion has resulted in their vulnerability to invasion by alien, i.e., exotic species. Among the exotic introductions, it is worth mentioning the impact of potted citrus plants imported from Madagascar, South Africa or Asia that unknowingly transported the vectors Diaphorina citri Kuwayama and Trioza erytreae Del Guercio together with their respective Asian and African strains of HLB. In 1968, local authorities signed a convention with the French Institute IRFA-CIRAD for rehabilitation of local citrus and fruit productions. Registered budwood, free of virus and virus-like graft-transmissible diseases, was imported from the INRA-CIRAD Station de Recherche Agrumicole SRA in San Giuliano, Corsica in 1969, and subsequently propagated within strict phytosanitary nursery requirements.

Fig. 1: Geographical location of Reunion Island.

Fig. 2: HLB bacteria in the sieve tube of a mandarin fruit columella (sample from St Pierre Reunion) TEM section courtesy of M. Garnier INRA Bordeaux (X12,000).

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Scion-rootstock combinations with tolerance to endemic Tristeza and Vein Enation Woody Gall were chosen because these two aphid-borne infectious diseases were the only ones known to occur naturally in Reunion, in addition to what would later be described as HLB. Unfortunately, this phytosanitary policy did not alone solve the problem of citrus dieback in Reunion, thus prompting new research efforts. With the advent of transmission electron microscopy (TEM), Laflèche and Bové (1970) were the first to identify the HLB causal agent in the sieve tubes of presumptively infected orange leaves originating from South Africa, India and Reunion Island. This necessitated that specific horticultural strategies had to be developed to address the difficult problem of a new graft-transmissible and psyllid -borne disease. With the exception of non-grafted seedlings of Madame Vinous orange, our HLB experimental work was conducted on the following clonal selections grafted on Troyer citrange: Clementine SRA 83, Dancy tangerine SRA 136, Valencia orange SRA 18, Pineapple orange SRA 141, Hamlin Orange SRA 41, Ortanique SRA141, and Orlando Tangelo SRA 21. Two other orange selections grafted on Rangpur Lime were also used: Sweet Seedling SRA 50 and Washington Navel SRA 141. Various other SRA selections were included in foliar diagnostic surveys linked to HLB evaluations in private orchards, among them Lee Page Minneola.

Ambient temperature conditions during HLB epidemics Experiments were conducted on the lowland coastal area up to 150m in elevation, with ambient conditions of temperature as follows: summer season (November to April) characterized by average monthly maximum temperature of 29.05 °C and average monthly minimum temperature of 20.8 °C, followed by the winter season (May to October) with average temperatures respectively 24.7 °C and 16.3 °C. Reunion supported the African and Asian strains of HLB with the two respective psyllid vectors. I used a thermograph to monitor in the screen-house of Bassin-Plat (120m above sea level) the total number of degree days, to estimate the total number of degree-hours above 30°C (DH/30) during the summer season, which always ranged below 50 hours. According to Schwarz and Green (1972), this value must reach 800 to 1500 hours to suppress symptom expression of the temperature-sensitive African strain of HLB, while Bové, et al. (1974) demonstrated that these symptoms disappeared under 27°C and 32°C

night and day temperatures, respectively. Within our experimental program, we could not discriminate any specific effect distinguishing the African or Asian HLB strains.

Fig. 3: HLB in Reunion A) 8-yr-old HLB-affected orchard in Le Gol, B) HLB-affected 8-yr-old orange tree, C) left: HLB-affected Ortanique fruit, right: healthy equivalent age fruit on symptomless twig, D) Trioza erytrea, E) Diaphorina citri.

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Assessment of HLB symptoms in the orchards of Reunion The term Huanglongbing (HLB) refers to a series of debilitating symptoms affecting citrus trees. The first noticeable sign of HLB contamination is a yellowing of foliage in sectors of the tree (originally described as yellow shoots). However, this symptom may not be specific strictly for HLB, and further observations of individual leaves are necessary to detect other early symptoms, “green islands against yellow background” or “blotchy mottle,” which are more indicative of HLB early infection (Figs. 4.2 and 4.3). Several months after these first symptoms appear, other symptoms can develop in subsequent weak flushes resembling nutrient disorders producing “green veins against yellow background” reminiscent of manganese deficiency may appear (Fig. 4.1). Leathery consistency of the lamina with vein corking

symptoms on the upper side of the leaf is another symptom that develops later (Fig. 4.5). At a later stage, upright small yellow leaves with symptoms similar to zinc deficiency are produced, followed by gradual premature leaf drop and twig dieback (Fig. 4.4). The phenology of affected trees is altered, with off-season new flush and flowering, followed by poor fruit set and by fruit drop soon after bloom. The fruits at the ripening stage show inverted coloration (initial color change at the peduncular end, Fig. 3C), a lopsided aspect, low sugar and juice contents, and aborted seeds. These fruit symptoms are common on both orange and mandarin trees affected by HLB6. Adequate disease evaluation is necessary to address the spatial and temporal complexity of these symptoms in infected orchards. The prevailing horticultural situation discussed, evolved from orchards established with healthy nursery trees and naturally contaminated by psyllids over the following years, in spite of conventional insecticide sprays. Under such conditions, the following methodology for HLB assessment was adopted: Select the proper time to scout for HLB spread, and return regularly during the same

season. For instance, in Reunion, the end of spring flush (mid October) was chosen in order to include newly matured fresh leaves for canopy observations.

Identify and record individual affected trees during each repeated scouting.

At each scouting visit, monitor the severity of HLB symptoms in the different sectors of the canopy of a given tree.

It was decided to use five categories of HLB severity corresponding to the five major components of the canopy as shown in Fig. 5: i. green twigs (supporting leaves and fruits), ii. fully lignified young branches of 10 to 20 mm diameter with grey colored bark (supporting green twigs), iii. sub-scaffold branches, iv. scaffold branches, and v. main branch directly connected to the trunk.

6 Inverted Fruit coloration and aborted seeds may also result from a contamination of the stubborn disease. All the SRA selection developed in Reunion were free of this disease

Fig. 4. Foliar symptoms linked to HLB in Reunion.

Fig. 5. Assessment of HLB symptoms on adult trees.

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The corresponding scale of symptom’s severity was rated from 0 to 5: 0: healthy aspect 1: scattered green twigs affected by yellow shoot 2: yellow shoot over an entire young branch

supporting several green twigs 3: yellowing over an entire sub-scaffold branch +

some twig dieback 4: yellowing over an entire scaffold branch + some

dieback of entire young branches 5: yellowing on all scaffold branches directly

supported by one side of the main trunk + dieback of entire subscaffold branches.

This rating 0 to 5 was given separately to eight canopy sectors of each of the adult trees monitored as shown in Fig 6. Although somewhat tedious, these HLB evaluations of each of the canopy sub-sectors were useful for assessing HLB epidemics in specific orchards of a given agro-climatic area, or for testing for tree recovery attained from antibiotic injections. Therefore, individual trees were evaluated on an HLB scale ranging from 0 to 40 with:

0 to 5 early foliage symptoms without any effect on yield. 5 to 15 phenological and physiological disorders inducing 50% yield reduction.

15 to 25 general yellowing of the canopy with entire sub-sectors affected by dieback, as shown on Fig. 3B, yield reduction of 80% compared to a healthy tree.

25 to 40 severe canopy leaf drop and dieback and halt to fruit production. During our experiments this canopy rating was applied for the following purposes:

i) monitoring the effect of antibiotic injections on a group of 36 trees planted in 1970 and canopy-rated during four successive years, 1977, 78, 79, and 80.

ii) assessing spatial and temporal epidemics on a group of 220 trees planted in 1970 and canopy-rated in 1977, 78, 79, 80

iii) evaluating the progress of epidemics in two groups of 1000 trees with group A planted in 1970 and canopy rated in 1975, 77 and 79; and group B planted in 1978 and evaluated in 1981-85.

2. Anatomical and physiological disorders attributed to HLB The term pathogenicity is used to describe the capacity of microbes (viruses, bacteria, fungi, protozoa) to produce infectious diseases in other organisms. Concerning citrus HLB, this term is not appropriate as long as Koch postulates are not fulfilled. In spite of this restriction, the anatomical and physiological disorders “attributed” to HLB, were investigated through various experiments conducted between 1978 and 1982 on container-grown young plants, or in affected citrus orchards, under the conditions prevailing in Reunion as described above. The following procedures were employed:

i) Histopathological examinations and physiological evaluation of graft-inoculated, container-grown, young orange Madame Vinous seedlings.

ii) Thorough horticultural and mineral assessment of 8-year-old, naturally HLB-infected oranges trees, that recovered after antibiotic injection.

iii) Additional data obtained from foliar diagnostic surveys on 8-year-old naturally infected groves.

In all cases, environmental conditions were identical and the dual-sampling methodology was used consisting of analysing healthy and HLB-affected individuals or healthy and affected twigs, as described above.

Fig. 6. Canopy evaluation method.

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3.1. Observations conducted on graft-inoculated orange seedlings Materials and Methods a) Blind midrib side graft on indicator plant Experimental transmissions of HLB by grafting were conducted to track phloem disorders and mineral deficiencies induced by the disease. Some 150 seedlings of Madame Vinous oranges (seeds originating from Willits and Newcomb nurseries, USA), were grown in 5-liter containers inside an insect-proof screenhouse in a mixture of peat moss and coarse volcanic sand. Seedlings were regularly supplied with liquid nutrient (Smith solution7) and used as indicator plants. At the age of 12 months, the seedlings were inoculated by inserting under the bark of their main stem, two pieces of leaf midrib with adjacent lamina, taken from affected adult source trees showing typical HLB symptoms. A total of 19 HLB-affected trees were individually identified in eight representative orchards by a painted number on the trunk. The intention was to develop a library of HLB local strains. Several of these source trees had tested positive for HLB by TEM observations of the leaf veins and fruit columella phloem (samples fixed with 4% glutaraldehyde in pH 7.4 phosphate buffer and dispatched to INRA-LBCM laboratory Bordeaux). Each HLB midrib source strain from a given candidate tree was grafted onto four indicator plants. Another group of seedlings was auto-inoculated with two pieces of healthy leaf midrib as a control. Saplings on which the graft had taken were pruned 40 days later as shown in the sequence in Fig. 7, and their re-growth observed for a period of 14 months. The technique illustrated on Fig. 7 was obtained from Vogel and described by Bove and Vogel (1981) who found the training of a single leader inoculated by side graft effective in transmitting both stubborn disease and HLB. b) Histopathological observations Regrowth leaves of 4 to 12 months old from graft-inoculated plants showing HLB symptoms and their equivalents on healthy plants were selected for two types of comparative histopathological observations

i) Central midrib transections were cut by hand with a razor blade and dipped for 5 minutes into 6% sodium hypochlorite, then rinsed with tap water and treated with a mixture of alum-carmine and iodine-green, which stained the cellulose membrane pink and the lignin green.

7 The Smith solution delivers the five indispensable metalloids N, P, S, B, and Cl, and the ten metals K, Mg, Ca, Na, Fe, Mn,

Zn, Cu, Mo in equilibrated proportions. The young orange seedlings were irrigated once a week with 0.1 to 0.5 liter of Smith formula according to the age of the plants, plus once a week with 0.2% ammonium sulphate solution. Smith formula:

Potassium nitrate 0.02 g/L Copper sulfate 4X10-6 g/L Calcium nitrate 0.08 g/L Zinc sulphate 4X10-5 g/L Magnesium nitrate 0.04 g/L Manganese chloride 0.1 g/L Ammonium sulphate 0.02 Boric acid 3X10-4 g/L Monosodium phosphate 3.3X10-3 g/L Ammonium molybdate 1.6X10-5 g/L Ferric sulfate 0.4X10-3 g/L

Fig. 7: a and b) leaf side graft, c and d) wrapping, e) untying & pruning.

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ii) Central midrib segments were fixed in glutaraldehyde and post-fixed with osmium, then dehydrated up to absolute alcohol and embedded in Epon-alraldite. Semi-thin sections obtained with an ultra-microtome were stained using parangon and then observed with light microscope following the technique of Salle (1981).

c) Thorough fresh weight/dry weight and mineral assessments After 32 months, three inoculated plants carrying the strain B14 from the orchard of Sucreries Bénard Le Gol were sacrificed for complete analysis. Three healthy control individuals were similarly sacrificed and analysed. Fresh plant tissues were weighed, separating leaves, green twigs, stem, tap root and feeder roots, then washed, and dried in oven at 60°C. The dry weight of separated organs was noted and their powders dispatched to the Laboratory of Plant Physiology IRFA-CIRAD Montpellier, where samples were mineralized dry at 520°C for analysis, all nutrients except nitrogen, boron, and chloride which required sulphuric acid digestion. The analysis was undertaken for 5 macronutrients (N, P ,K, Ca, and Mg) and 7 micro-nutrients (Na, Cl, Fe, Mn, Zn Cu, B, and Al. Results

a) Blind midrib grafting Table 1. Source trees selected for HLB blind bark grafting and results of successive inoculations.

Cultivar/rootstock

Field label

Age

Canopy rating

Treatment Antibiotic therapy

No. of successful

grafts Le Gol Etang Salé O. Pineapple/ Troyer B 14 6 years 25/40 None 3 / 4* O. Pineapple/ Troyer K 4 6 years 25/40 None 2 / 4 O. Pineapple/ Troyer A16 6 years 25/40 None 1 / 4 O. Valencia /Toyer D9 6 years 25/40 None 2 / 4 O. Hamlin/Troyer I 6 6 years 10/40 Tetracycline* 2 / 4 O. Hamlin/ Troyer E 16 6 years 15/40 Penicillin** 2 / 4 O. Valencia/Troyer E 9 6 years 5/40 Tetracycline* 0 / 4 O. Valencia/Troyer E 9 6 years 5/40 Tetracycline* 0 / 4 O. Sweet Seedling/Troyer B 12 6 years 5/40 Tetracycline* 0 / 4 O. Sweet Seedling/Troyer B 15 6 years 5/ 40 Tetracycline* 0 / 4 Rossolin Entre-Deux Clementine/Troyer A1 3 years 10/40 None 1 / 4 Dancy/Troyer A2 10 years 15/40 Tetracycline* 2 / 4 Turbot Petite Ile O. Hamlin / Troyer B 2 3 years 25/40 None 1 / 4 O. Hamlin / Troyer C 4 3 years 25/40 None 2 / 4 IRFA Bassin-Plat Tangelo Orlando/Troyer D 8 4 years 15/40 None 2 / 4 O. Hamlin / Troyer M 4 4 years 15/40 None 3 / 4 Tampon 17° km O. Sweet seedling A 1 15 years 20 / 40 None 1 / 4 O. Sweet seedling A 2 15 years 20 / 40 None 3 / 4 IRFA Petite Plaine O. Hamlin / Poncirus C 1 4 years 10/40 None 0 / 4

* The three successfully inoculated plants were sampled for dry weight/fresh weight plus mineral assessment (see Figs. 15a, 15b, and Table 2 below).

* Source tree injected with tetracycline solution 10 months before choosing a leaf for inoculum showing mild HLB blotchy mottling

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** Source tree injected with penicillin solution 10 months before choosing a leaf for inoculum showing mild HLB blotchy mottling

From a total of 76 inoculated plants, 27 developed HLB symptoms; i.e., 31% of transmission. The inoculations took place during the last two weeks of May 1979 at the beginning of the winter season, and the first symptoms appeared over the following 4 to 6 months, on the newly emerging shoots. After the production of two to three healthy-looking leaves, subsequent leaves developed manganese-like deficiency symptoms (Fig. 4.1) or more often with blotchy mottle (Figs. 4.2 and 4.3 ), which were then followed by smaller leaves showing zinc-like deficiency (Fig. 4.4). With time, some affected leaves also developed vein-corking symptoms on the upper side of the lamina (Figs 4.5). Details concerning each candidate source tree are presented on Table 1. b) Histopathology The midrib of healthy citrus leaves protrudes more on the lower than the upper side of the lamina, with the vascular bundle sections as seen on Figs. 8a and 9. With the development of HLB symptoms on the leaves, the central midrib gradually protrudes from the upper side of the lamina as a result of excessive phloem tissue produced by the intensive cambial activity, Fig. 8b. The final stage is the occasional appearance of vein-corking symptoms as shown on Fig. 8c and Fig. 10. The lower side of the midrib becomes concave, and the upper side becomes convex. The intense lignin deposit hardens the sclerenchyma layer such that

sustained cambial activity pushes the thickened phloem upwards. The upper side of the vascular bundle then pushes through the disrupted

epidermis, and a healing cork layer develops near the remaining xylem islands. However, the vein corking symptom is not unique to HLB,

Fig. 8. Schematic aspect of central midrib trans- section of orange leaves with progressive HLB symptoms.

Fig. 9. Partial aspect of lower side of healthy midvein transsection with spongy mesophyl, sclerenchyma cells phloem, xylem and medullar tissue.

Fig. 10. Transection of HLB-affected midvein at the stage of vein corking. Excessive phloem, proliferation is pushing the xylem upwards, and pushing out the upper part of the vascular bundle. Scl=Schlerenchyma Phy=Phloem Su=Cork layer.

Fig. 11: (Left) Hamlin orange leaf with low boron content 9 ppm, (Middle) Tahiti lime affected by a severe strain of tristeza virus, (Right) small HLB-affected leaf of Madam Vinous orange.

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since acute boron deficiency or severe stress from tristeza in lime may also induce vein corking as presented in Fig 11. Semi-thin cross-sections of healthy, newly mature leaves displayed vascular bundles of primary origin with a dormant cambium layer. The phloem exhibits regular distribution of large diameter sieve tubes with adjacent companion cells, apparently to facilitate the source-sink translocation of carbohydrates and related metabolites. The cell wall of the sclerenchyma remains rather thin (Fig. 12a). Conversely, the HLB blotchy mottle equivalent infected leaves display a sclerenchyma layer with denser lignin deposits (Fig 12b). In addition, premature senescence of primary sieve tubes and companion cells near this hardened sclerenchyma layer, is characterized by numerous osmiophilic dots and thicker cellulose cell wall. The cambial

layer becomes active, producing secondary phloem, which is then “squeezed” against the xylem, reducing the diameter of new sieve tubes. Ultimately, hyperplasia of phloem tissue terminates with a vein-corking syndrome as described above on hand razor transections (Fig. 13). Considering that sieve elements are known to be extremely sensitive to injury, the cambium’s reaction in HLB-affected leaves is probably the result of the shorter lifespan of sieve tube functionality, inducing phloem

hyperplasia. Careful TEM observations of ultra-thin sections showed callous occlusions plugging the sieve plate pores often associated with the presence of the HLB organism as presented in Fig 14.

Fig. 12. Semi-thin transversal sections of healthy midrib (A) and HLB blotchy-mottle midrib (B). Xy-xylem, Ca-cambium, Ph-phloem, Si-schlerenchyma, Th-thickening of cell wall, and P-proliferation of phloem tissue. (Red cellulosic cell wall, Green lignified cell wall).

Fig. 13. Hyperplasia linked to HLB vein-corking.

Fig. 14. Trans-section of a sieve plate, with apparent plugging of sieve pores, associated with HLB organisms (x12 000) Courtesy M Garnier

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c) Fresh weight/dry weight and mineral analyses

Fig. 15a shows a young seedling of Madame Vinous Orange 13½ months after midrib graft inoculation with HLB (strain B14 Pineapple Orange on Troyer citrange from Le Gol orchard), as compared to the equivalent control plant, Fig. 15 1, while vigorous flushing with green leaves was obtained on the three control seedlings. In all, during the 15 ½ months after the date of inoculation, the control plants

produced some 60 healthy leaves. The HLB strain B14-inoculated plants began to produce 2 to 3 healthy looking leaves followed by an average of 10 leaves with blotchy mottling, and later by 25 to 30 tiny yellowish leaves of only 1.5 to 2 cm length. Fig. 15b shows the sequence of this leaf production. Sacrificed healthy plants, and HLB strain B14-inoculated plants were weighed, separating their respective leaves, green twigs, trunks, main tap roots and feeder roots to obtain fresh and dry data. The results are presented in Table. 2.

Fresh weight: Compared to the healthy control plants, the HLB strain B14-inoculated plants produced 78% less fresh leaves, 79 % less fresh green twigs, and 83% less feeder roots. Concerning the trunk and tap root, the drop of fresh weight was only 24 and 31%, respectively. Dry weight: Among the dry weights of the trunks and tap roots, there were no significant differences since the growth diameter over a period of only 15 ½ months is too low, even for healthy control plants. However, for leaves, green twigs, and feeder roots, the reduction of dry weight was 66%, 72% , and 81%, respectively.

Fig. 15a. Two-year-old Mme Vinous Orange seedlings 1) Healthy control plant, 2) HLB inoculated plant with the strain B14. Pineapple Orange Le Gol.

Fig. 15b. Details of the two plants appearing on Fig 14 with Plant 1 healthy control, and Plant 2 graft inoculated HLB B14 strain.

Table 2. Global fresh/dry weight assessment of HLB-inoculated Madame Vinous Orange with B 14 strain versus healthy ones (in grams).

Control healthy orange seedling HLB inoculated orange seedling Plant No.1

Plant No.2

Plant No.3 Average

Plant No.1

Plant No.2

Plant No.3 Average

FW DW FW DW FW DW FW DW %DM FW DW FW DW FW DW FW DW %DM

Leaves 20.7 5.4 15.2 3.4 19.0 5.5 18.3 4.8 28.2 4.8 1.9 3.6 1.7 3.4 1.4 3.9 1.6 42.8

Twigs 13.2 3.5 6.6 1.8 13.6 4.6 11.1 3.3 29.7 2.7 1.0 2.3 1.1 1.8 0.8 2.2 0.9 42.7

Trunk 30.4 15.0 43.2 21.5 48.1 18.5 40.5 18.3 45.2 37.5 22.4 29.2 19.9 25.0 15.3 30.5 18.8 61.8

Taproot 22.7 11.9 20.7 9.1 28.3 13.7 25.5 11.5 45.1 17.0 8.3 17.5 11.0 17.9 14.1 17.4 11.1 63.7Feeder roots 28.9 7.6 21.4 5.7 17.1 4.1 22.4 5.8 25.8 2.2 0.8 5.9 1.5 3.2 0.9 3.7 1.0 28.9FW = Fresh weight, DW = Dry weight, %DM= % of dry matter

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Clearly, the HLB strain B14 was associated with a sharp reduction of green biomass output (leaves, young fruits, and green twigs and rootlets), the most active organs for production/accumulation of carbohydrates and mineral uptake. d) Mineral analyses Tissues of plant organs from three healthy and three HLB strain B14-inoculated plants were collected, dried, and ground to a powder. Results of these preparations were analyzed for their mineral contents (Table 3). Five elements were worthy of mention. The major

consequence is the lower calcium content in the HLB-affected leaves. Surprisingly, the zinc and manganese leaf contents are very similar for both groups of plants, although visual deficiency symptoms were noticed for the HLB-inoculated plants. The latter accumulate iron and zinc in the feeder roots, and aluminium in the leaves.

Dry weight/Fresh weight and mineral assessment of 10 years old orange trees

Materials and Methods Here also, the dual-sampling methodology consisting of analysing healthy and HLB-affected individuals, or healthy and affected twigs on the same tree, were used.

Table 3. Mineral contents for 5 elements comparing healthy plants (H) and HLB strain B14 inoculated plant (D). % dry matter ppm dry matter Plant Organ Ca Fe Zn Mn Al

Leaves H 2.260 216 61 21 109 D 1.270 281 76 19 231

Green Twigs H 1.0 309 42 14 85 D 0.6 153 47 11 92

Trunk H 1.31 223 77 12 137 D 1.23 140 32 109

Tap root H 0.93 805 59 27 406 D 0.59 512 95 24 242

Feeder roots H 0.81 7,735 643 432 695 D 0.74 9,335 1,090 464 934

Table 4. Chronology of two HLB orange trees submitted to antibiotic therapy Chronology Tree I4 Tree D4 Plantation Planted in 1970 Planted 1970 September 1977 HLB Canopy rating 22/40 Canopy rating 18/40 October 1977 Pruning + 2kg NPK fertilizer* Pruning + 2kg NPK fertilizer* December 1977 Injection 18g penicillin** Injection 6g tetracycline*** April 1978 Harvest 18 kg Harvest 24 kg September 1978 HLB Canopy rating 10/40 HLB Canopy rating 3/40 October 1978 18g penicillin + 2kg NPK 6g tetracycline + 2kg NPK April 1979 Harvest 33kg Harvest 130 kg September 1979 Canopy rating 31/40 Canopy rating 6/40 October 1979 18g penicillin + 2kg NPK 6g tetracycline + 2kg NPK February 1980 Harvest 4kg Harvest 78kg

March 1980 Uprooting for global assessment

Uprooting for global assessment

* Combined 15, N 15%, P205 12%, K20 24%. ** Injection of 18g diluted into 6 liters of water. *** Injection of 6g diluted in 2 liters of water.

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Two orange trees (Pineapple SRA 141 grafted on Troyer citrange) were selected in the orchard Suceries Bénard Le Gol for their similar HLB canopy rating. The history of these individuals is presented on Table 4. Tree I 4 reacted positively to a first penicillin injection (see details of injection in Fig. 16), but relapsed considerably afterwards in spite of two additional injections. Its cumulative yields of 1978, ’79, ‘80 amounted to 55kg. Tree D4, treated by tetracycline, recovered notably and gave cumulative yields amounting to 232 kg. The two trees were uprooted in March 1980 and submitted to the same global evaluations as presented above for the young seedlings Madame Vinous orange. Both trees are shown in Fig. 17 just before and after their uprooting in March 1980. The I4 tree branches were separated into mildly, severely or very severely affected ratings to check for the presence of fluorescent gentisic-glucoside following the technique of Feldman and Hanks (1969).

Soil analysis The private orchard (Le Gol Benard Ltd), consisting of 1 ha planted with 220 oranges trees originating from the SRA and grafted on Troyer citrange, was leased for 4 years for research purposes. Although planted in 1970 with SRA disease-free material, and established in the middle of sugar cane

blocks with preventive insecticide sprays, these orange trees were naturally inoculated via psyllid at an early stage of planting. The sandy basaltic soil was randomly sampled ( 8 points combined with three soil layers each) and submitted for soil analysis. Results

The physical and chemical characteristics of the soil are presented in Table 5. After three successive penicillin injections, tree I4 displayed a weak canopy and had a

Fig. 16. Pressurized 5Kg/cm² bottle for antibiotic trunk injection.

Fig. 17. (Upper left) Tree I4 HLB canopy rated 31/40 produced 4 kg of fruits. (Upper right) Tree D4 HLB canopy rated 6/40 produced 78kg of fruits in two sacks. (Bottom) Both trees were prepared for fresh weight/dry weight and mineral assessments. (March 1980).

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global fresh weight of 78 kg including a small fruit crop of 4 kg. Whereas, the equivalent healthy-looking, tetracycline-injected tree D4 has an overall fresh weight of 174 kg including a fruit crop of 78 kg (harvested 1 month before maturity). No remarkable results were noted for presence of gentysic glucoside. The branches of tree I4 tree were combined for the fresh weight/dry weight and mineral assessments. The details of fresh and dry weights as well as percent dry matter content were broken down into 21 different organs or parts and are presented on Table 6. Table 5. Physical and chemical characteristics of Le Gol (Benard Ltd) orchard (20m asl), 1 ha, 220 trees.

% Granulometry Organic matter and N Soil absorbing complex* ppm Micronutrients

Clay Silt Fine

sands Coarse sands

Org. Mat. C

Total N C/N

ppm P Ca Mg K Na CEC pH Zn Mn Mo

SO4 Soluble

1 5.7 13.8 23.9 56.7 2 1.17 1.02 1.1 109 7.7 4.9 1.02 0.46 17.7 6.5 14 124 14 164

2 5.7 12.3 24.2 57.6 1.72 1. 0 0.9 1.1 94 6.1 5.3 0.84 0.49 12.3 7.2 17 95 21 194

3 6.8 15.1 29.3 48.6 1. 22 0.7 0.59 1.2 51 5.4 5.5 0.60 0.43 11.8 7.4 20 94 18 162

1) Soil layer 0-10 cm, 2) 10-30 cm, 3 ) 30-60 cm. Average of random 8 samplings per soil layer *milli-equvalents/100g of soil. N.B. Total dry biomass was deducted from representative fresh samples taken on each type of organs.

For fresh leaf biomass, tree D4 had a total of 22 kg compared to only 4.8 kg for the HLB-affected symptomatic tree I4. Leaf dry weights gave similar differences with a factor of 5 between healthy and affected individuals. Similar results were also obtained for green twigs (Table 6). The fresh biomass differences for feeder roots were lower than expected, although the HLB tree I4 displayed a larger proportion of brownish rootlets. The weight of fresh bark branches is generally half as much as for tree I4. The corresponding global mineral contents of each tree for five macro-nutrients and eight micronutrients are presented on Table 7. Red lines indicate mineral deficiencies, and green lines indicate mineral accumulation. Clear mineral deficiencies in Ca, Zn, and Mn were found for the leaf lamina.

Table 6. Fresh /dry weights and % dry matter for trees I4 and D4 broken down into 21 parts plant organs.

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Table 8. Mineral nutrients immobilized and/or exported by TREES I4 and D4 and equivalent amounts of commercial fertilizers (in g).

Immobilized amount in the trees

Exported amount as fruits total

of three harvests

Immobilized (I) + exported

(E) equivalent commercial fertilizer + exported D4 I4 D4 I4 D4 I4 D4 I4 N 498 226 515 157 1013 383 Ammonium sulphate 20% N 5060 1900

P 66 30 122 24 188 54 Tricalcique phosphate 46% P2O5

940 270

K 310 113 526 144 836 257 Potassium sulphate 48% K2O

1530 470

Ca 682 260 233 62 915 322 Slake lime C03Ca 1700 600 Mg 87 36 58 16 145 52 Magnesia MgO 290 88Fe 18.1 6.7 5.6 1.4 23.5 8.1 Iron sulphate 7H20 109 38Mn 0.6 0.7 0.3 - 9 0.7 Manganese sulphate 4H2O 3.6 2.8 Zn 0.8 0.3 0.6 - 1.4 0.3 Zinc sulphate 7 H20 6.4 1.4 Cu 0.7 0.2 0.3 - 1.0 0.2 Copper sulphate 7 H20 4.0 0.8 Bo 1.1 0.4 - - 1.6 0.4 Borax 11% B 14 3.5

Deficiencies were recorded in the lamina of the tree I4 for Ca, Fe, Mn and Zn. The K and Ca contents of tree I4 feeder roots was half of that of tree D4 feeder roots. The global fertilizer efficiency was calculated for the trees I4 and D4 by integrating the equivalent of exported minerals for the 1978, 1979 and 1980 yields on the one hand, and for the immobilized mineral portion represented by the tree itself on the other hand. The data are

presented in Table 8 for five macro-nutrients and five micro-nutrients. The data presented on Table 8 are indicative of economic losses resulting from trees seriously affected by HLB and left unremoved in commercial citrus orchards.

Table 7. Corresponding total mineral contents for macro and micro-nutrients.

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3.3 Foliar diagnostic surveys on 8-year-old naturally infected groves Materials and Methods Four private orchards in Reunion, that had been planted in 1970, were sampled in December 1978 for leaf mineral diagnostics. Individual trees selected for their similar HLB canopy ratings were submitted to dual leaf sampling methodology by choosing canopy sectors with healthy-looking twigs and twigs with HLB symptoms (yellow shoot with blotchy mottling).

For each sector, batches of 30 leaves belonging to the ranks N° 3 and N° 4, as shown in Figure 18 A and B, were collected separately. Leaf samplings were carried out on fruiting or non-fruiting twigs of newly emerged flushes. Leaf batches were washed under tap water, then dried in a oven at 60°C, weighed and sent for analyses of the five macro-nutrients N, P, K, Ca, and Mg, and the eight micro-nutrients Na, Cl, Fe, Mn, Zn, Cu, B, and Al. These surveys were conducted in three orchards planted with Clementin SRA 63 grafted on Troyer citrange, and one orchard planted with Valencia Late, SRA 18, grafted on Troyer. In the second orchard, antibiotic-injected trees were chosen for leaf sampling.

Results Clementin SRA 63 in three orchards Since the only mineral content differences between healthy leaves and HLB symptomatic leaves were for Ca, Mn, and Zn, the results presented below only list these elements.

Compared to healthy-looking canopy sectors, the mineral contents of HLB-symptomatic sectors were 50 % lower for calcium and zinc, and 39% lower for manganese, thus falling below the deficiency threshold for manganese of 20 ppm indicated ( Chapman 1960). The deficiency in Mn was solved by foliar sprays of 250 ppm manganese sulphate. Valencia Late SRA 18 (Le Gol orchard) A group of 9 orange trees selected in the orchard Le Gol (Sugar estate Benard, Ltd) were combined into three triplets of individuals presenting the same canopy rating. Here again, the

Fig 18: Ranks of leaf samplings on fruiting and non fruiting twigs with: A) HLB symptoms, and B) healthy looking twigs.

A

B

Table 9: Mineral contents of batches of 30 leaves of healthy and HLB symptomatic (Clementine SRA 63/Troyer citrange)

Orchards No.

trees HLB Canopy

rating Type of

twigs Symptom

aspect % dry

weight Ca ppm Mn ppm Zn

Vitry Petite Ile (300m asl)

3 5/40 to 8 /40fruiting twigs

healthy 3.35 52 34 blotchy mottle

0.94 34 16

Amplis Saint- Benoit (150m asl)

2 12/40 to 18/40fruiting twigs

healthy 3.13 28 31 blotchy mottle

1.46 15 24

Badre Tampon (400m asl)

4 20/40 to 25/40fruiting twigs

healthy 4.70 36 29 blotchy mottle

2.94 22 16

All tests were from Clementine on Troyer. The results obtained from 9 trees belonging to 3 different orchards are presented.

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two elements Ca and Zn showed the sharpest differences between healthy and HLB-symptomatic leaves, as presented on Table 10.

The two control trees H16 and J7 with severe canopy rating of 32/40, were of particular interest because they presented few surviving symptomless twigs for which Ca and Zn leaf contents were significantly higher than the equivalent leaf contents on HLB-symptomatic trees. 3.3 Outer trunk wood mineral analysis on 4, 10 and 18-year-old trees. Considering that citrus HLB is found associated with phloem transport problems, the question was raised if xylem disorders could be detected also in local orchards of Reunion. The possible occurrence of blight, a disease associated with xylem plugging and plant wilt, was of particular interest and investigated with the following procedures:

i) Visual checks for wilting of the entire canopy, ii) Water trunk injection testing, iii) Zinc content of outer trunk wood chips collected with a forestry auger (8 samples

per trunk after the removal of the bark ), The blight disease was reported from Florida, Brazil, and South Africa generally on 10-year-old adult trees with a higher rate of occurrence on Rough Lemon scion-rootstock combinations. The characteristic global canopy wilting prior to leaf fall was not reported from Reunion, and the numerous injections of antibiotics went smoothly. In addition, one of the peculiarities of blight is the accumulation of zinc in the outer trunk wood (Wutscher, et al. 1977) together with a slight increase in nitrogen, potassium (Williams and Albrigo, 1984). Twenty trees in four different HLB-contaminated orchards of Reunion were observed, analysed, and compared with four HLB adult trees from Indonesia, and five blight-affected trees from South Africa and Florida (Lee et al. 1984; Wutcher et al. 1977).

Table 10. Comparative foliar diagnostics for Ca and Zn contents for three triplets orange trees.

Triplets orange trees

Tree No.

Canopy rating Sept. 1977

Canopy rating Sept 1978

Type of twigs

Leaf Symptoms

%Ca Dry

Matter

ppm Zn Dry

Matter

Control H16 31/40 32/40 non fruiting HLB 1.70 25

Healthy 2.10 32 Penicillin 18g G20 27/40 4/40 fruiting Healthy 3.11 34Tetracyclin 6g E9 27/40 9/40 fruiting Healthy 3.20 37

Control E15 9/40 16/40 fruiting HLB 1.85 14

Healthy 3.27 44

Penicillin 18g G19 12/40 10/40 fruiting HLB 2.70 26

Healthy 3.22 43 Tetracyclin 6g F10 15/40 2/40 fruiting Healthy 4.02 50

Control J7 20/40 32/40 non fruiting HLB 1.41 22

Healthy 3.64 40 Penicillin 18g H9 26/40 3/40 fruiting Healthy 4.04 47 Tetracyclin 6g J 9 19/40 3/40 fruiting Healthy 3.89 42

NB. Leaf mineral diagnostic performed in December 1978, and two previous antibiotic injections in Sept. 1977 and Sept. 1978.

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From the above results, no evidence could be found of any zinc accumulation in the outer trunk wood of HLB-affected adult trees sampled in Reunion and Indonesia. Ortanique, SRA 110, and Valencia Orange, SRA 18 trees displayed low manganese contents. No global canopy wilting typical of blight was detected in Reunion and Indonesia nor any specific water trunk injection difficulties. So far, South Africa seems to be the only country known to host both diseases, HLB and blight.

3. General discussion HLB infectiousness was demonstrated by blind midrib side-graft on orange Madame Vinous indicator seedlings. A phloem surface contact of some ½ mm² between healthy orange bark and infected midrib was enough to induce acute HLB symptoms. About 6 months after such inoculations, typical leaf mottling symptoms developed under the environmental conditions prevailing in Reunion Island. Vein phloem degeneration mentioned in Indonesia by Tirtawidjadja, et al (1964) and by Schneider (1964) in South Africa, were clearly observed after experimental graft transmissions of HLB. Semi-thin sections observed by light microscope have shown that

Table 11. Outer trunk wood mineral contents comparing HLB and Blight affected trees. HLB Reunion Tree No. Canopy Rating Zn ppm % N % K Mn ppm B ppm

HLB Reunion Ortanique SRA 110/Troyer 4yr 1 0/40* 4.3 .4 0.13 1.8 23 2 0/40* 3.1 0.4 0.13 2.5 20 3 0/40* 2.3 0.4 0.10 1.8 15 4 30/40 1.4 0.4 0.28 1.2 19 5 28/40 2.1 0.4 0.18 1.2 19 6 32/40 1.2 0.4 0.13 0.6 15 Valencia Or. SRA 18/Carrizo 10 yr 1 0/40 1.0 0.3 10 2.0 17 2 25/40 2.4 0.4 0.28 1.2 18 3 25/40 2.7 0.4 0.21 2.5 17 4 32/40 2.2 0.4 0.12 5 28/40 2.3 Clementin SRA 63/Rough lem.18 yr 1 0/40* 4. 0 0.35 0.13 6.0 2 0/40* 4. 0 0.38 0.13 4.0 3 35/40 3. 0 0.41 0.14 2.0 4 20/40 5. 0 0.35 0.17 2.0 5 20/40 8. 0 0.41 0.14 6.0 Or. Thompson /Rough Lemon 18 yr 1 10/40* 2.0 0.35 0.11 2.0 2 20/40 6. 0 0.33 0.10 3.0 4 25/40 7. 0 0.35 0.10 7.0 5 30/40 5. 0 - 0. 12 6.0 HLB Indonesia Orange tree 1 0/40** 1.2 - 0.10 2 15/40 1.2 - 0.16 3 20/40 1.7 - 0.14 4 30/40 4.7 - 0.24 Blight South Africa(Lee et al. 1984) Valencia L. /Rough lemon N. Transval 1 1. 8/4 16.3 2 1.6/4 29.1 3 2.2/4 17.0 Blight Florida (Wutscher et al. 1977) 1 ¾ 25.0 0.54 0.27 6.2 12 2 ¾ 17.0 0.47 0.21 5.0 11 * Treated by tetracycline injections. ** Isolated tree in upland area.

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young HLB-contaminated leaves produced secondary phloem, with a tendency for phloem proliferation as increasing infection severity develops. Normal sieve tubes-companions cell arrangements in the leaf vascular bundles are disturbed, with reduced diameter transections of sieve tubes. Swollen and degenerated phloem tissue is squeezed between a strongly lignified sclerenchyma layer and the xylem. As a result, the phloem is gradually rejected through the torn epidermis of the upper side of the lamina. Numerous osmiophil dots are indicative of biochemical changes of the sieve tube contents. On ultra-thin tangential sections observed by transmission electron microscopy, sieve plate pore plugs were observed in association with the presence of HLB bacterial organisms proliferating within the cytoplasmic elaborated sap. On HLB-affected plants, the leaf biomass production is considerably reduced together with that of the feeder root biomass. Infected plants have lower capacities for water and mineral nutrient uptake. On adult trees, the appearance of sectored canopy mineral deficiencies with corresponding gradual desiccated green and woody organs, including fruits and seeds, is ensuing. The ultimate syndrome is the sectored dieback. As opposed to strict physiological mineral deficiencies that show up within the entire canopies, HLB-driven zinc and manganese deficiencies always display in sectors. In individual leaves, there is a non-symmetrical appearance of blotchy mottling in the lamina; within the canopy, a non-symmetrical appearance of yellow shoots; and finally patchy dieback in the branches. This asymmetry, including lopsided fruits, clearly discriminates the consequences resulting from a disturbed phloem situation from that of a general xylem plugging. Tetracycline injections and, to a lesser extent, penicillin were able to rid the foliage of these symptoms. In terms of chemical assessments, the sharp reductions of leaf calcium content for both young and adult plants must be considered as one of the most important aspects of HLB-induced physiological disorders. Calcium plays a major role at the level of plant cell wall and is probably linked to sieve pores plate occlusions. Calcium-driven protein bodies seem to be responsible for sieve pore occlusions, disrupting the translocation of phloem sap. For the above reasons, we believe that HLB may affect the cytoplasmic continuity between adjacent sieve tube elements, especially the functionality of sieve pores. Sieve tubes are composed of living enucleated cells functioning in close synergy with adjacent companion cells for the active source-sink translocation of carbohydrates and relative metabolites. This is precisely where HLB pathogenicity seems to operate.

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Part Two HLB etiological status of differential antibiotic therapy

Psylla sap feeding mouth construction Atractivity of Rutaceous plants for T. erytrea and D. citri

1. HLB etiology status in Reunion

Considering the tedious and costly diagnostic procedure of sampling trees and assaying leaf midribs or fruit columella via transmission electron microscopy TEM (samples being dispatched to INRA Bordeaux in 2% buffered glutaraldehyde for sieve tube examination on ultra-thin sections), a field experiment was initiated to check if the HLB citrus disease could be ascribed to a bacterial origin. For this purpose, the curative effects of two antibiotics were tested on naturally contaminated trees in open orchards exposed to insect vector transmissions. Material and Method Antibiotic injections were tested in a 6-year-old orange grove (one hectare) leased from a private sugar estate (Sucreries Bénard du Gol). Although planted in 1970 with SRA disease free material, and established in the middle of sugar cane blocks with preventive insecticide sprays, these orange trees were naturally inoculated via psyllids at an early stage after planting. The trial was comprised of eleven triplets of trees displaying the same HLB canopy rating. The triplets, comprising a total of 33 trees, were injected once a year during the main flush season over a 3-year period. (October 1977, 1978, and 1979) and with penicillin, tetracycline or plain water, respectively. Canopy rating was assessed once a year following the main flush, and fruit were harvested for yields and weights just before full maturity to avoid yield losses due to theft. The individual tree dosage of penicillin was 100mg per kg of fresh weight; i.e., for 6-year-old trees, an injection of 18g was diluted into 6 liters of water. For tetracycline, 35mg per kg of fresh weight was chosen; i.e., 6g diluted into 2 liters of water. The plot design of the field experiment is presented in Figure 19. The data concerning the soil fertility of this orchard are presented in Table 5. Three of these triplets were used also for mineral leaf diagnostics (see Table 10). The pressure for the injections was 5kg/cm² (see the aspect of pressurized bottles in Figure 16 and the details described in Aubert (1979). Following penicillin injection, the concentration of antibiotic was monitored by antibiogram assays of crushed leaf midrib segments. The extracted liquid was deposited on 6mm blotting Schleicher-Schull mini-discs No. 2668. The discs were previously placed on Petri dishes containing a Grove and Randall

Fig. 19. Plot design of the 11 orange triplets (details Table 12).

Fig. 20. Penicillin assays obtained wthe midribs of injected and non injetrees.

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medium sown with ATCC 9341 Sarcina lutea. Leaf midrib penicillin concentrations were estimated by measuring the diameter of S. lutea growth inhibition around the mini-discs (Figure 20). Results Penicillin concentration in midribs.

No phytotoxic effect was noticed with penicillin, while tetracycline induced brief willow-leaf symptoms prior to stimulating vigorous healthy looking re-growths (Figure 21). Table 12. Detailed results of differential antibiotic injections on HLB affected trees

Concerning the results of penicillin antibiograms performed on crushed leaf-midribs, it was found that detectable bacteriostatic effects could last up to for 120 hours after the onset of tree injections (Fig. 22). Detailed observations of canopy ratings and corresponding yields for the 33 trees are presented on Table 12 for the period between September 1977 and March 1981. The statistical interpretations of this field experiment confirmed the following

Fig. 21. Temporary phytotoxic effect due to tetracycline injections of 6g per a tree followed by vigorous regrowths.

Fig. 22. Penicillin midrib contents versus time in hours after the onset of injections of 3, 6, 9 and 12g respectively in liquid solution, or 15g in the form of a gel. Triangle =control injected with tap water.

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points: i. a significant but short-term canopy recovery was followed by substantial yield increase with penicillin injections, and ii. the beneficial effect of tetracycline outperformed penicillin for both canopy recovery and yield improvement as shown on Figures 23 and 24.

By combining all the data relative to canopy ratings for December 1978 with the corresponding harvests of March 1979 and the canopy ratings of December 1979 with the corresponding harvests of March 1980, it was possible to fit a regression curve for the relationship of visual HLB symptom severity and fruit loss. This curve computed from a total of 66 observations (3 treatments X 11 replicates X 2 years) is characterized by a sharp negative exponential relationship across the interval of 5 to 15 canopy ratings. Discussion The tree recovery obtained with penicillin treatments was indicative of the HLB disease origin. As penicillin is known to hinder the formation of peptidoglycane layers of the bacterial cell walls, the HLB plant pathogenic organism is a probable Gracillicute Gram negative prokaryotic organism (following the classification of Gibbons and Murray 1979). A conclusive demonstration of this was reported by Garnier, et al (1984) on graft-inoculated young orange seedlings treated by root drenching with penicillin, and submitted to fine TEM observations of their phloem-restricted HLB bacterial cell wall layers.

Fig. 23. Effect of antibiotic injections on canopy recovery. Average canopy ratings of 13 injected trees.

Fig. 24. Effect of antibiotic injections on yields. Average yields of 13 injected trees.

Fig. 25. Fitted negative exponential regression line of the relationship between fruit yield and canopy rating 0/40.

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But neither penicillin nor tetracycline resulted in long-term suppression of HLB, probably due to the bacteriostatic rather than bactericidal effect of antibiotics and also to possible developments of resistance. This experiment demonstrated how rapidly treated trees relapsed following the second and third penicillin injections, a clear indication of the virulent character of the HLB organism. Mild canopy visual symptoms were clearly associated with serious loss of fruit production as shown on Figure 25. Tetracyclin injections were considered for some time as a possible control strategy to potentially mitigate the citrus HLB problem. The technique of syringe injecting high concentrations of N pyrrolidinomethyl tetracycline (PMT) at a rate of 1.5 g in 25 ml of water, proposed by Buitendag and Bronkhorst (1984), was tested in Reunion for three years (1985, 1986 and 1987), on a group of 9 triplets of adults mandarin trees. Although easier to accomplish, this technique was found less effective than the pressurized bottle device that injected 4 times more tetracycline HCL that was diluted in a volume of water 80 times greater. Pressurized bottles outperformed the yield recovery by 45% as compared to the syringe PMT technique. Therefore, antibiotic therapy was not as effective at controlling HLB disease as originally expected, probably because the bacteriostatic effects of antibiotics in the phloem tissue elicited an auto-immune system as it does in animals.

2. Mechanisms of psylla sap feeding Adults of Trioza erytrea and Diaphorina citri reared in an insectarium were collected in feeding position by gently cutting the leaves and depositing them in a freezer at -15°C for five minutes. Individual insects were then isolated by cutting out a small piece of their corresponding leaf substrate and dipped while still in feeding position into a solution of 2% buffered glutaraldehyde. They were subsequently treated by the Laboratoire d’Accueil by Scanning Electron Microscopy CNRS Paris, and prepared following the technique of Guillaumin (1980). The lateral views of Figures 26 and 27 show the details of T.eryreae and D. citri heads, with a larger organ of sight for the former, but similar leaf sucking mouthparts in ventral position at the junction of the

Fig 26 Head of Trioza erytreae. Fig 27 Head of Diaphorina citri. 1) Vertex 2) Conical front head 3) Labium and protruding stylet (x 120).

Fig. 28. Stylet bundle penetrating Fig. 29. Distal part of the stylet. near a stomata. (x 1200)

B . A u b e r t , P a g e | 24

head and thorax for both insects. The labium encloses a stylet which is used to pierce and suck the fluid from a leaf. Three telescoping segments forming an internal labial groove surround and support the stylet bundle which is approximately 4 microns in diameter. The stylet bundle is covered by a setal sheath of saliva that seems to accumulate at the surface of the epidermis (Figures 28 and 29). The three threads may slide jerkily against each other for piercing the leaf tissue up to the phloem. Gustatory sensillas mediate acceptance or rejection of plant fluid thus playing a major role in locating tissue feeding.

3. Attractivity of Rutaceous plants Container-grown 15-month-old seedling plants belonging to 20 species of the 8 following genera: Citrus, Fortunella, Poncirus, Triphasia, Vepris, Toddalia and Microcitrus, were placed into environmental control conditions to expose gravid females of either D.citri or T. erytreae. For the Asian psylla, the ambient conditions used for rearing were natural daylight with 34°C,

and 50% relative humidity during the day, and 22°C and 85% relative humidity during the night. For the African psylla, rearing was carried out in an air-conditioned chamber with 23° C, 80% relative humidity, and 40w artificial OSRAN light by day, and 16°C and 100% relative humidity by night. The feeding, oviposition and nymphal behaviors, were observed over a period of 50 days in both cases. The results are presented in Table 13.

Fig. 30. Head of the ventral face of Diaphorina citri. 1) Labium, 2) Apex of labium, 3) Stylet, 4) Clypeus, 5) Conical front-head, and 6) Coxa. (x 270)

Table 13. Differential host plant relationships of T. erytreae andD. citri.

* Behavior described by ZHAO (in Barkley, et al 1979).

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Part three Environmental and social constraints

Integrated vector management for the control of HLB epidemics In collaboration with J. Etienne INRA Guadeloupe

Intertropical islands, used often as natural quarantine facilities over the previous centuries, were and still are critical steps for the spread of vector-borne citrus diseases such as HLB. Biocontrol approaches of integrated vector management combined with targeted preventive actions were found relevant for sustainable sanitation in Reunion. HLB and the socioeconomic situation of Reunion Island Distinctive conditions for HLB epidemics. The densely populated territory of La Reunion (currently ~800.000 inhabitants, with a large proportion of migrants from the rim of the Indian Ocean and China), and adjacent Mauritius alike, were facing uncontrolled conditions for HLB epidemics (Moreira 1967 Bové and Cassin 1968, Catling 1973). The reasons were: i. presence of the African vector Trioza erytreae thriving without any of its natural parasitoids, and of the Asian vector Diaphorina citri that was poorly controlled by a single endoparasitic wasp species Diaphorencyrtus aligarhensis; ii. A great diversity of climates over a small volcanic island of only 2500 sq.km with steep topography combined with tropical tradewinds and occasional hurricanes, thus offering multiple opportunities for vectors to build up and HLB disease to appear; iii. Substantial colonies of ornamental and wild Rutaceous plants harboring the two vectors; iv Fragmented land ownership of commercial citrus orchards interspersed with countless small citrus plantings and backyard trees; v. Lack of expedient diagnostic tools to discriminate HLB infection from physiological disorders; and vi. Limited research funding from the agricultural research institution IRFA-IRAT-CIRAD in charge locally. A patrimonial citrus production likely to exacerbate HLB problem In Reunion, rainfall, relative humidity and temperature greatly influenced citrus psylla population upsurges, with T. erytreae more adapted to high-lying windward cool and wet areas, and D. citri preferring dry, hot leeward low-lying areas. Depending on the season, overlap of psyllid territories was possible. Furthermore, the intermixing of both types of HLB organisms (i.e., African HLB showing symptoms only in cool climates as opposed to Asian HLB that is less temperature dependant) was enhanced by the capacity of each citrus psyllid to transmit either pathogen. Not surprisingly, individual trees were found to host both

Fig. 31 The ultra-peripheral regions of the E U. Fig. 32. Map of average yearly rainfall in Reunion Fig. 33. Map of altitude gradients Fig. 34 Map of commercial citrus orchards 2002. (courtesy D. Vincenot Chambre d’Agriculutre de la Réunion).

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strains of agents Ca. Liberibacter asiaticus and Ca. Liberibacter africanus simultaneously (Garnier, et al. 1996). Taking advantage of the ecological situations, local growers had eagerly cultivated a multitude of citrus types, from deeply colored mandarins in the mountains, to limes and grapefruits in the coastal areas. This diverse range of commercial plantings was closely intertwined and formed a continuum with small citrus gardens, backyard trees, and urban hedges of Murraya paniculata (L.) Jack var. Exotica, orange jessamine. The citrus map, Figure 34, shows the present commercial orchards in Reunion, [400ha producing 10.000t of fruit, (Insa et al. 2002)] and traces back the struggling situation of the early 1970s when a mere output of 800t was obtained. At that time, tree declines due to HLB epidemics were typically compensated by accelerated cycles of orchard replanting with unsafe nursery material. Under such conditions, any attempt of compulsory eradication for citrus and citrus relatives in Reunion appeared socially and technically infeasible. Strategies of sustainable sanitation based on integrated vector management (IVC) In line with the recommendations of the International Organization of Citrus Virologists (IOCV), especially during a post-conference tour taking place after the 6th congress held in Mbabane, Swaziland 1972, and initiated by J. M. Bové, the following strategy was decided: Incentives for replanting certified disease-free material: Registered budwood, free of

graft-transmissible diseases, was received from SRA San Giuliano, Corsica in 1969. New cultivars, especially easy peelers, exhibiting attractive qualities were propagated within strict registered nursery requirements (up to 35.000 trees/year). To enhance the removal of affected commercial orchards, local authorities (Chambre d’Agriculture de la Réunion) refunded the cost of replanting and trained extension service personnel to survey/assist a new generation of growers aware of the benefits of preventive sprays. Although uncertain in term of prognosis, this strategy was nevertheless considered more appropriate than a costly and hazardous eradication scheme. Such a propagation system of certified planting material, first initiated in 1969, is still operating currently, with modern insect-proof and fully covered greenhouses delivering virus-free and citrus-canker-free planting material. This compulsory CAC- EU label: conformité agricole communautaire, is now required since Reunion is an ultra-peripheral region of the European Union.

Fig. 5: Tullus, Ltd container-grown citrus nursery in Reunion (courtesy M. Roux Cuvelier CIRAD Réunion Sept. 2008).

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3. Biological control

Biological control of the vectors. Concurrently, a specific biological control program was launched in 1974. Its aim was the introduction, rearing, and mass release of primary parasites of psyllid nymphs (Order Hymenoptera superfamily Chalcidoidea,). Primary ectoparasitic wasps Tamarixia sp were found much more efficient than primary endoparasitic Psyllaephagus sp or

Diaphorencyrtus sp. Therefore, emphasis was put on the African Tamarixia dryi, which originated from Nelspruit, South Africa, and on the Asian Tamarixia radiata, which originated from Badal, West Punjab, India. For the former wasp, 33,000 adults were released on the island, thus corresponding to 50 adults per square kilometer of citrus area; and this was followed by a release of 3,500 adults in the restricted citrus area of the dry lowlands where D. citri was predominant. A careful elimination of secondary or tertiary parasitoids enabled these wasps to establish properly and resulted in a drastic reduction of vector populations within 3 years (Etienne and Aubert 1980).

The two primary ectoparasitic

wasps have similar biologies and show remarkable host-searching ability. The females lay eggs on psyllid nymphs of the 3rd, 4th and 5th instars. Their lifecycle is only 12 to 14 days as opposed to 21 days for endoparasitic wasps. Being ectoparasitic, the larvae feed on and suck out the hemolymph of the psyllid nymphs. Adults pupate in the mummies of the nymphs and emerge by chewing a hole through the thorax of the psyllid host. The size and shape of the hole, and the meconium left by the parasitoid in the nymph mummy give a signature of the absence of secondary or tertiary parasitoids. Taxonomic studies related to this bio-control were carried out with the assistance

Fig. 36. Map of wasps releases for the biological control of both psyllids .Releases took place preferably within non-sprayed backyard trees.

Fig. 37. Parasitoid complex of T. erytreae nymphs after Catling 1969, Mc Daniel and Moran 1973 .Tamarixia sp shows a characteristic white patch on the abdomen.

Fig. 38 Mummy of D. citri nymph with exit hole of emerged T. radiata adult.

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of the Museums of Natural History of Pretoria and London (Annecke et al 1971, Prinsloo 1981 and Hollis 1984) and with information on hyperparasites available from Husein and Nath (1924). The primary endoparasitic wasp, Psyllaephagus pulvinatus, imported into Reunion from South Africa, was largely outcompeted by T. dryi and disappeared. Trioza erytreae, easily spotted by its remarkable formation of galls left by feeding nymphs on citrus leaves, survived for six years on semi-wild lemon plants at 900m elevation in an extremely wet area, but was eventually completely eliminated. The only explanation for this unexpected result is the build-up of T. dryi on an alternative polyphagous psyllid, Trioza litseae (Hollis 1984), occasionally feeding on avocados, citrus, vanilla and papaws, but predominantly on a common aromatic shrub Litsea glutinosa. This plant, which originated from Asia and Australia, was imported into Reunion many years ago, and was largely disseminated by local birds. T. dryi ex T. litseae was found conspecific with T. dryi ex T. erytreae (Prinsloo unpublished). In Eastern and Southern Africa, T. dryii is apparently unable to parasitize any psyllid other than T. erytreae. Neither T. litseae nor Litsea chinensis occur in Africa, both having an Asian origin. Relative to the Asian citrus psyllid, D. citri, it survived on pruned hedges of Murraya exotica but was rarely seen on citrus, even on neglected trees. T. radiata was unable to parasitize any psyllid other than D. citri (Aubert and Quilici 1984), and further laboratory studies were conducted for disclosing its basic life-history traits (Fauvergue and Qulici 1991, Quilici 1992).

Epidemiological survey Temporal increase of HLB disease was evaluated by visual

canopy assessment of two groups of commercial orchards of 1000 trees each, with the assistance of trained extension service personnel. Group A of orchards was planted in 1970, 1971 before the biological control program, and evaluated in 1975, 1977 1979 and 1980, respectively. Group B planted in 1977, 1978 after the biocontrol program of psyllid vectors was similarly evaluated respectively in 1981, 1984. Comparative rates of spread of the disease were evaluated following Van der Plank’s analysis. In group A, 50% of the trees were commercially lost seven years after planting, while in group B, disease rate was so low that 50% loss would theoretically occur around 2015. Gottwald recently reviewed the epidemiology of HLB by comparing Reunion, Chinese, Brazilian, and Florida situations (Gottwald, et al, 2007).

Conclusion on the Integrated Vector Management story of Reunion In spite of an initial massive psylla build-up and spread of the pathogen, substantial results for controlling the HLB disease were obtained in a rather short time for a problem affecting perennial plants. The training of extension service consultants for monitoring the disease and helping/educating the farmers was an important aspect of the program. As stressed by Ohmart, integrated management cannot succeed without the awareness of the growers (Ohmart, 2008). The new generation of orchards sprayed with horticultural oil to control other insects and mites resulted in an extremely low rate of disease progression. A common

Fig. 39. Temporal increase of HLB epidemics within two groups of orchards planted before or after the biological control of both vectors

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practice adopted by the farmers in commercial orchards and by many residential owners of small gardens and backyard trees was to voluntary remove and replant affected trees. Today HLB is a forgotten nightmare in Reunion to the point that any research on this dangerous disease is being discontinued. However, accurately detecting the last HLB foci for their

eradication would be wise. Neither, hurricanes nor the strange episode of rural hyper epidemics of human‘chikungunya’ virus disease transmitted by A. albopictus in 2006, had any marked effect on the new citrus psyllid ecosystem, notwithstanding massive emergency applications of fenithrothion and deltametrine to eliminate the adult mosquito vectors of this human disease. The major challenge for the citrus growers now is the replacement of old healthy trees established over the past 30 years, with novel cultivar selections that have better prices on the local fresh fruit market. With as much as 130 Corine biotopes and

a rich flora including 600 endemic species and some 2400 imported/introduced species, Reunion is considered a hot spot of world biodiversity. This may explain the success of the psyllid vector biocontrol. Similar results were obtained in the neighboring Mauritius Island, where the Reunion fidings were duplicated concomitantly. Integrated Vector Management in Guadeloupe The first detection of D.citri in the Caribbean islands occurred in Guadeloupe on backyard orange trees in January 1998 (Étienne, et al., 1998). After one year of investigation, no parasitism was found on these intrusive D.citri colonies, and the introduction of Tamarixia radiata from Réunion was conducted in January 1999 (Étienne, et al., 2001). The rearing and release of a few hundred wasps did succeed to establish preventively the ectoparasitoid and to reduce notably D. citri populations not only on citrus backyard trees and Murraya exotica hedges, but also over the 360ha of local commercial orchards of limes, oranges and mandarins. This program of biocontrol was developed together with three others targeting citrus pests that arrived suddenly in the late 1990s and for which specific wasps were also imported: Anagyrus kamali against the hibiscus pink mealybug, Ageniaspis citricola against the citrus the leaf miner, and Lysiphlebus sp against the brown citrus aphid. In April 1998, Guadeloupe had sent a warning to the entomology department of UF-IFAS Homestead; and three months later, D.citri was soon detected in South Florida (Knapp, et al., 1998). Today, ten years after the preventive biocontrol launched against D.citri in Guadeloupe, the HLB organism has not yet been detected there, and the neighboring Martinique and La Dominique islands are still free of D. citri. In the greater Antilles, the Asian vector of HLB was found in Cuba in 1999, then in Haiti in 2000, in the Dominican Republic in 2001, in Puerto Rico in 2002, and in Jamaica in 2003 (Halbert and Nunez, 2004). T. radiata population imported from Reunion and reared in Guadeloupe develops a sex ratio of 65 to 70% females. It is speculated that this is tied to a complex of endosymbionts, especially Wolbachia, known to induce overriding chromosomal sex determination, as

Fig. 40. Twenty five-year-old citrus orchard in Reunion (Courtesy Vincenot Chambre d’Agriculture de la Réunion 2008)

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indicated on the new T. radiata population of Florida imported from Taiwan (Meyer and Hoy 2008). Incidentally, the origin of Taiwanese T. radiata population comes from a group of 33 females and 20 males dispatched from Réunion between February 1984 and May 1986 (Chiu, et al., 1988). In addition to Florida, T. radiata was also reported from Cuba and Puerto Rico. It has to be seen what will result in terms of vector control and HLB spread for these territories, depending on local ecosystems, specific ectoparasitoid introductions, and strategies of integrated management.

Other recent island situations The accidental introduction of Trioza erytreae in Madeira in 1994 (Fernandes and Franquinho, 2001) and its extension some years later into Tenerife, Gomera and Palma Canary Islands (Perez, Padron and Hernandez, 2002), is a new threat for the citrus production of the western part of the Mediterranean Basin. Similarly, the presence of D. citri on the islands of Hawaii and Maui (Conant, et al., 2007) and the interception in 2008 of the Asian HLB vector on ornamental Rutaceous plants (curry plant) dispatched from there to California is an additional example of the need for relevant surveys and controls on island situations. In Asia, D. citri was recently reported from Ryu Kyu, the southern-most island group of Japan stretching towards Taiwan (Khono, et al., 2000) while in the Asia Pacific region, the arc of Indonesian islands and Papua New Guinea represent a threat for northern Australia (Beattie and Barkley, 2008). Conclusion The sudden spread of HLB in citrus-producing areas previously regarded as HLB-free highlights the potential threat of one of the most serious diseases of citrus. Considering the extreme fertility of both psyllid vectors with each female laying as many as 1000 to 2000 eggs in a matter of some weeks, chemical protection alone may end in a vicious cycle with rising levels of resistance and damage to the environment. Boosting carefully screened natural enemies and helping farmers to learn the dynamics of their ecosystems may offer interesting alternatives. At any rate, insecticide sprays must target seasonal flushes, especially those synchronized with hot and dry periods when D. citri upsurges are likely to take place. Acknowledgements This multidisciplinary research work on HLB would not have not been achieved without the scientific and technical support of J.M. Bové, R. Vogel, M. Garnier (plant pathologists), D. Annecke, ECG Bedford, J. Etienne, S. Quilici (entomologists), J. Marchal (plant physiologist), D. Guillaumin and G. Salle (insect & plant anatomists), and T. Gottwald (epidemiologist). Laboratory and screenhouse equipment, orchard facilities, labor force, and grants for numerous trips outside Reunion were made available with the help of the successive IRFA-CIRAD chief officers J. Cuillé, J. Charpentier, E. Laville, J. Letorey, B. Moreau and JP.Gaillard who all recognized the serious threat of HLB. The participation to IOCV conferences of Athens 1975, Mildura 1979, Iguazu 1983, Valencia 1986 and to the ISC conference of São Paulo 1984, were important for strengthening our HLB motivation. Finally the local grower’s association, Chambre d’Agriculture de la Réunion, was instrumental in surveying/eradicating HLB affected orchards with the active participation of extension service officers especially L. Picard, S.Sabine, J. Ethève, D. Vincenot, G. Rossolin ,

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and in promoting the availability of disease free citrus planting material through registered citrus nurseries.

A prolongation of this success story was the launching of the UNDP-FAO regional project RAS/86/022 with the outcome of the IOCV Fuzhou conference (1995), where the official common name for the disease ‘Huanglongbing’ was adopted instead of greening in recognition of the pioneering work of Lin Kung Hsiang.

Thank you finally for the IOCV newsletter.

Warm regards,

Bernard

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