South American Leaf Blight of the Rubber Tree (Hevea spp.): New

INVITED REVIEW

South American Leaf Blight of the Rubber Tree (Hevea spp.): New Stepsin Plant Domestication using Physiological Features and

Molecular Markers

REINHARD LIEBEREI*

Biocenter Klein Flottbek and Botanical Garden, University of Hamburg, Unit of Applied Ecology and Crop Science,Ohnhorststr. 16, D-22609 Hamburg, Germany

Received: 23 February 2007 Returned for revision: 16 March 2007 Accepted: 25 April 2007 Published electronically: 23 July 2007

† Background Rubber trees (Hevea spp.) are perennial crops of Amazonian origin that have been spread over thewhole tropical belt to guarantee worldwide production of natural rubber. This crop plant has found its place inmany national economies of producing countries, and although its domestication by selection of suitable genotypeswas very slow, it contributes a lot to the welfare of small farmers worldwide. Its development is limited by severediseases. In South America, the main fungal disease of rubber trees is the South American leaf blight (SALB)caused by the ascomycete Microcyclus ulei. This fungus inhibits natural rubber production on a commercialscale in South and Central America.† Scope The disease is still restricted to its continent of origin, but its potential to be distributed around the worldrises with every transcontinental airline connection that directly links tropical regions. The need to develop controlmeasures against the disease is an urgent task and must be carried out on an international scale. All control efforts sofar taken since 1910 have ended in a miserable failure. Even the use of modern systemic fungicides and use ofgreatly improved application techniques have failed to prevent large losses and dieback of trees. The results ofresearch dealing with both the disease and the pathosystem over more than 50 years are summarized and placedinto perspective.† Future Prospects A detailed knowledge of this host–pathogen combination requires understanding of thedynamics of Hevea leaf development, the biochemical potential for cyanide liberation, and molecular data forseveral types of resistance factors. Resolution of the Hevea–SALB problem may serve as a model for futurehost–pathogen studies of perennial plants requiring a holistic approach.

Key words: Hevea, South American leaf blight, resistance factors, source–sink relationship, stage-specific resistance,cyanogenesis, defence, resistance screening, scopoletin, marker-assisted breeding.

INTRODUCTION

Natural rubber (NR) produced from latex of the rubber treeHevea brasiliensis nowadays accounts for about 40 % ofthe world’s total rubber consumption; 60 % is delivered bysynthetic processes. The annual world demand for naturalrubber is constantly growing because of its typical physi-cochemical properties, which are still not achieved bysynthetic products. Worldwide production reached 9.7million tons in 2004 (FAO, 2006). Most plantation areasare located in South East Asia, especially in Thailand,Indonesia and Malaysia, with growing production areas inVietnam and China. In contrast to this geographical distri-bution of successful production of natural rubber, the biologi-cal centre of origin of the rubber tree is the Amazon basin inSouth America. The annual production of natural rubber inthis vast area of Brazil is only 97 000 t, which is about 1 %of world production and far below the national needs ofBrazil for rubber. The reason for this discrepancy is the factthat natural rubber in Asia, India and Africa can be grownon plantation scale, while in Brazil natural rubber is almostcompletely produced in extractive production systems inthe Amazon basin, i.e. in co-operatives that collect latexfrom wild-growing rubber trees (e.g. Dean, 1987).Plantations in Central and South America so far have never

reached full production because they are destroyed beforethe trees reach physiological maturity (Table 1).

The main reason for low production and failure of plan-tation growth is South American leaf blight (SALB), arubber tree disease caused by the endemic fungusMicrocyclus ulei, an ascosmycete that together with itshost plant originates from the Amazon area. Since thebeginning of rubber exploitation in the region, all attemptsto plant rubber trees at a plantation scale in South anCentral America have failed completely (Table 1), and todate all control measures against this disease have beenunsuccessful (Smith et al., 1995). Neither chemical protec-tion nor biological control measures have promoted thedevelopment of healthy and productive plantations (Chee,1980). Breeding and selection (Goncalves et al., 1983;Gasparotto et al., 1997) as well as agronomical measureshave not led to successful production on a plantationscale. The last concentrated efforts were taken in the gov-ernmental programs PROBOR I, II and III of Brazil from1966 to 1989 (Smith et al., 1995).

At this point in time, the fungus has not spread to othercontinents on which rubber is cultivated, at least in partbecause of intensive control of international air traffic andfreight shipments from South America to other tropicalareas. At present, there are almost no direct intercontinentalflights between Brazil and other humid tropical countries,* Forcorrespondence. E-mail: [email protected]

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but future direct intercontinental flights are a probability.Disease control is becoming more complex, and becauseof these factors the probability of an uncontrolled spreadof this pathogen is high.

It has been demonstrated that the majority of cultivatednatural rubber tree genotypes outside Brazil are susceptibleto the disease (Chee, 1976). This finding, from both inten-sive field tests and laboratory studies, is not surprising.Rather, if one considers the way rubber tree seeds were dis-tributed historically, the fact that the pathogen has neverbeen introduced into the Old World is remarkable. Thecomplete genetic basis for rubber production outsideBrazil can be ascribed to only some hundred genotypes,all based on the Wickham collection. Wickham broughtmore than 80 000 rubber tree seeds from the Amazonregion to Kew Gardens. Only a small number of these recal-citrant seeds survived the transport and storage and germi-nated at Kew. These seedlings formed the basis of the‘oriental’ clones on which the whole plantation cultureoutside the Americas has been based. Some new introduc-tions to the South East Asian production areas were madelate in the 1980s, as a result of the first international approachto dealing with the rubber tree germplasm problem.

The introduction of Microcyclus ulei into the South EastAsian areas of production is a continuing threat; such anintroduction would effect a drastic change in the nationaleconomies of the rubber-producing countries as well asthe world market for all fine rubber products, especiallyfor high quality tires for aeroplanes and automobiles(Chee and Holliday, 1986). The serious nature of this threatto the world economy has recently led to the decision toinclude South American leaf blight in a list of biologicalweapons (United Nations Office on Drugs and Crime,

http://www.unodc.org/unodc/terrorism_weapons_mass_destruction_page005.html).

In this paper, the state of knowledge concerning thespecific nature of the plant–fungus interaction is analysedwith an intent to elucidate the bottlenecks that limit thespread of the pathogen and to highlight factors that needto be given special consideration for the successful futuredevelopment of natural rubber germplasm. My goal inthis review is to facilitate production of plant materialtolerant or resistant to M. ulei and to initiate the develop-ment of an international management strategy for successfulresistance.

DESCRIPTION OF THE HOSTPLANT MATERIAL

The genus Hevea, family Euporiaceae, comprises elevenspecies (Goncalves et al., 1983). The main source forhigh quality natural rubber is Hevea brasiliensis, a treethat contains latex vessels in the inner bark and that canbe tapped by cutting the bark. Latex flow begins immedi-ately after injury to the bark and stops when internalreactions in the latex cause coagulation of this milky sub-stance. Fused isoprene droplets form a plug in the laticifers.The quality of a rubber tree is correlated with the latex com-position and production, most importantly the flow rate oflatex after cutting. Selection of trees with superior latexquality has been successful, but almost all of these highlyproductive trees are also highly susceptible to attack bySALB. Hevea brasiliensis is a tall tree native to theAmazon basin, that may reach heights of more than 20 mwithin a forest, with a trunk of up to more than 50 cm indiameter at 1.5 m stem height. Since the beginning of thelast century there have been numerous attempts to cultivatethis tree in plantations throughout the tropics. This specieshas not been completely domesticated, but selection ofuseful parameters has been made with respect to latexquality, tapping suitability and stress resistance. Concen-trated selection, such as that which has occurred in the dom-estication of main world crop plants like wheat, maize orpotato, remains to be carried out with this technicallyuseful plant.

The first major steps towards scientifically rational rubberplant development can be linked to the International RubberExcursion for collection of germplasm material in 1984(Allen, 1984; Bahia et al., 1985). Many subsequent geneticimprovement programs for natural rubber now pursuehigh latex quality, reduced panel dryness, different canopyforms, good root formation, annual leaf shedding patternand reflushing of the canopy, etc. (e.g. CIRAD, 2005).However, agricultural and agroforestry breeding researchleading to understanding and control of SALB, or manage-ment of resistance to it, is still not a focus of research anddevelopment activities.

There is no mass production of latex in Brazil; rubber isonly obtained from small plantations in areas on themargins of the ranges of the forests, such as in MatoGrosso del Norte, where environmental conditions areunfavourable for fungal development (Gasparotto et al.,1997). In the Amazon region, latex is still extracted from

TABLE 1. Impact of South American leaf blight on rubbertree cultivation in Central and South America

Surinam 1911 40 000 trees planted1918 Plantation destroyed, first reported epidemics

by M. uleiBrazil 1927 Fordlandia: 3 200 ha, 200 000 trees

1933 Plantation destroyed by M. ulei attack1936 Belterra: 6 478 ha1943 Plantation destroyed by M. ulei

Panama 1935 Plantation created by Goodyear Comp.1941 Plantation destroyed

Colombia 1941 Epidemic development of M. uleiCosta Rica 1942 Epidemic development of M. uleiBrazil 1967 Start of SUDHEVEA, a three-step-programme

for the development of rubber cultivation inCentral Amazonia

1972–1975 PROBOR I1978–1982 PROBOR II1984–1994 PROBOR III1986 Programme was stopped: about 100 000 ha of

the 150 000 ha of plantation were alreadydevastated

Brazil 2002 Scientific meeting about new efforts towardsrubber cultivation in areas affected by SALB

Brazil 2005 Plantations only in areas free the fungalpressure: in all other areas latex is collectedfrom wild-growing rubber

Colombia 2006 Programme on selection of rubber treesresistant to SALB

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wild-grown trees in the same manner as it was 100 yearsago. Rubber trees in the primary forest occur in low abun-dance, about two trees per hectare. For the related speciesHevea guianensis, a density of 1.8 trees (.10 cm at1.50 m stem height) per hectare has been reported(Degen, 1999). Rubber gatherers can normally collectlatex from 100–120 trees per day. This number of treesconstitutes a ‘seringal’, a tapping trail in the rain forest.

Hevea brasiliensis, a perennial tree that can have 50 yearsof commercial life, has a distinctive juvenile stage in whichthe growth and developmental pattern is quite distinct fromthat of adult trees (Halle and Martin, 1968). The growingrubber seedling is characterized by a rhythmic-flushgrowth pattern. Whorls of leaves in successive cycles areproduced throughout the year. These whorls are separatedfrom each other by pronounced elongation of the internodesfollowing the formation of a new whorl. Each whorl con-sists of 9–15 leaves, inserted in a 3/8 spiral phyllotaxypattern. This process leads to rapid development of thin,but elongated, stems. After 5 or 6 years the crown developsthe first lateral branches, and successive whorl formation isreplaced by annual leaf shedding. Each year, the entirecanopy is shed at the beginning of the dry season. Refolia-tion occurs at the beginning of the new rainy season (Cheeand Holliday, 1986). Like many other members of theEuphorbiaceae, rubber tree inflorescences are made up ofmale, female and bisexual flowers (Dornelas and Rodrı-guez, 2005). Pollination is primarily dependent on smallinsects such as midges and thrips, but self-fertilizationand inbreeding occurs to various degrees. Self-incom-patibility has been observed and established by experiment(Goncalves, 1986), but male-sterile clones (such as GT-1)also occur (Goncalves, 1986). In recent breeding projects,cross-pollination or outcrossing has been shown to lead tobetter fruit set (Hamzah et al., 2002). Unfertilized pistillateflowers quickly wilt and die (Hamzah et al., 2002). Theseeds produced in the three-lobed fruit capsules are liber-ated by explosive dehiscence, a mechanical ‘explosion’ ofthe fruit shell. Seeds are short-lived and loose their poten-tial to germinate within 2 months of storage underambient conditions (Holliday, 1970).

THE FUNGUS: BIOLOGY ANDRACE STRUCTURE

The fungus is known by several names. The teleomorph ofthis fungus is Microcyclus ulei (Henn.) von Arx, a memberof the bitunicate ascomycetes. Synonyms are Dothidellaulei Henn. 1904 and Melanopsammopsis ulei (Henn.)Stahel 1917. The conidial state is Fusicladium macro-sporum Kuyper 1912 and the pycnidial state isAposphaeria ulei Henn. A thorough account of the fungalsystematics is given by Chee and Holliday (1986). In1962, Muller and von Arx revised the systematics ofthe fungus and transferred the genus Dothidella toMicrocyclus. An examination of the morphology and anupdated taxonomic description of this species, documentedwith excellent illustrations, has appeared in print (Chee andHolliday, 1986).

The first report of M. ulei on wild Hevea spp. was madeby Ule (1905), who collected samples in 1900 and 1901from Brazil near the river Jurua and in 1902 from Perunear Iquitos. In subsequent years, outbreaks of severeinfection in densely planted plots of H. brasiliensisoccurred in many places in South and Central America(Table 1). In all cases, the plantations were destroyedwhen the trees reached the maturation stage between 6 to7 years and changed from rhythmic-flush growth toannual leaf-shedding patterns. Belgrave (1922) and Weir(1929) gave early warnings of the extreme danger ofSALB to rubber plantations, especially those in SouthEast Asia.

Mass production of conidiospores and the rapid build-upof inoculum is the causal step in the epidemiologicaldevelopment of fungal infection in flushing canopies. Theconidia are short lived, whereas the ascospores producedin black stromatic structures survive for several months.These ascospores can initiate new infection cycles as soonas susceptible young leaf tissues are produced by the host(Chee and Holliday, 1986). It is important to note that theinitial infection in flushing tree canopies often starts fromconidia that are present throughout the year on young leafstages of rhythmic flushes of the seedlings or young,freshly budded trees.

The pathogen can be grown on complex media (Chee,1978; Junqueira, 1985). Sexual stages do not appear to beformed in vitro, but mass production of conidia can beinduced by synchronization of spore formation using appro-priate light conditions (e.g. Lieberei et al., 1983; Junqueiraet al., 1988; Rivano, 1992). New research activities invol-ving examination of fungal races and virulence variationhave been undertaken.

Mattos et al. (2003) recently reported the results ofstudies based on 12 rubber plant clones. The differen-tiation set of rubber tree genotypes used by theseauthors for race identification was virtually based on thegenotypes selected for the studies of Junqueira et al.(1988). Thirty-six profiles of virulence have been ident-ified among fungal isolates. Host ranges and quantitativeranges of aggressiveness of the isolates varied strongly.None of the isolates was able to infect all host genotypes.These results were combined with previous screening dataof Junqueira et al. (1988), Rivano (1997), Hashim andAlmeida (1987) and Chee (1976) and, in summary, atotal of 68 races with different host reaction spectra havenow been identified. The susceptible host-genotype rangeis very large.

Furthermore, the physiological tolerance of the isolates totemperature and relative humidity, as well as to complexenvironmental factors (Gasparotto et al., 1989a, b, 1991),suggests that the potential for adaptation of fungal isolatesto new environments is high. This variability underscoresthe potential threat of this fungus to new rubber growingareas outside South and Central America.

Important steps for analysis of the diversity of the fungusand its genetic polymorphism have also been carried outusing molecular approaches (Le Guen et al., 2004). Infungal isolates from Brazil and from French Guiana, micro-satellite markers were described and assessed with respect

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to their variability within the isolates. This approach offersa way to group the highly variable complex isolate groupson a genetic information basis.

THE DISEASE: PLANT REACTIONS TOFUNGAL ATTACK

Microcyclus ulei attacks young leaves, stems or fruittissue of five or more species within the genus Hevea(H. brasiliensis, H. benthamiana, H. spruceana,H. guianensis, H. camporum; Chee and Holliday, 1986).Early breeding concepts for resistance were based on theobservation that, in addition to highly susceptible geno-types, plants also occurred that seemed to be immune orcompletely resistant to M. ulei (Bos and McIndoe, 1965;Goncalves, 1968; Chee, 1976). These resistant plants wereused in crossings with high-yielding individuals of the‘oriental’ (Wickham) clones. Unfortunately, most of theoffspring of these crossings were susceptible to at leastsome of the fungal strains, and with progressive identifi-cation of the fungal virulence sprectrum it became clearthat the concept of specific or complete resistance sensuParlevliet (1975) was not applicable to the rubber tree.Subsequent research was redirected to identify factors thatcontribute to general resistance (Simmonds, 1989). Theseconsist of many interactive factors in the pathogen–hostplant relationship that minimize disease development.Important studies have addressed the physiologicalaspects of resistance with the aim of identifying processesinvolved in the plant–fungus interaction (Hashim et al.,1980; Hashim and Pereira, 1989; Mevenkamp, 1992;Garcıa et al., 1995a, b) and, finally, to develop biochemicalmarkers for marker-assisted breeding programs. Based on abroad range of observations, a comprehensive view of manyof the various stages of the plant–fungus interaction hasbeen achieved. Junqueira et al. (1988) prepared detaileddescriptions of lesion types produced by a diverse set ofhost genotypes. Combinations of various host genotypeswith selected fungal isolates varying in virulence spectraled to the formation of a sequence of lesion types withgraded differences in the occurrence of necrotic tissue,lesion size, number of spores formed, distribution of coni-dial layers in the lesion, and stroma formation as a latesymptom (Figs 1 and 2). Five main developmental levelsin the host–pathogen interaction, which together contributeto the formation of phenotype resistance, were identified(Giesemann, 1986; Junqueira et al., 1988).

(1) Germination and pre-penetration phase with earlyimpact of the plant on the spores’ infection potential.

(2) Penetration and initial infection phase with induction ofearly resistance responses.

(3) Leaf colonization, intercellular growth and ramificationof hyphae; a phase that is considerably influenced bycell wall modifications and leaf maturation processes.

(4) Formation of conidiophores, which leads to differencesin the spore number and spore distribution in andaround the lesion.

(5) Stroma production and ascospore formation.

Quantitative variations of each of these were observed;they ultimately combine to produce the phenotype of in-complete or partial resistance. In addition to these highlyvariable pathosystem features, there exists a stage-specificresistance. Only young, developing leaves (Fig. 1A), imma-ture fruits and young stems of this tree species are suscep-tible. When infection pressure is low and leaves are notinfected during the young stages of ontogenetic develop-ment, they develop to highly resistant leaf stages. Only afew studies have addressed these various aspects of physio-logical resistance (Blasquez and Owen, 1957; Junqueiraet al., 1990).

Germination and hyphae formation, pre-penetration phase

When the conidiospores of M. ulei come into contactwith young leaf tissues of susceptible genotypes, thespores germinate on the reticulate surface of the rubbertree leaves (Fig. 1B–E). Once in contact with a wettedleaf surface, the spores germinate and are not washed offby rain, as they are tightly affixed to the cuticle by anelectron-dense substance (Fig. 1E) (Giesemann, 1986).Infection hyphae penetrate the epidermal layer of thelower leaf side (Fig. 1F) in the contact zone of three epider-mal cells with or without formation of appressoria. Thedegree of formation of branched germ tubes and the for-mation of appressioria is dependent on interaction in thepre-penetration phase. (Hashim et al., 1978; Giesemann,1986).

Spore germination and germ-tube formation occurimmediately after contact of the conidiospores with theleaf. The morphology of the germ tube varies with thereticulate leaf surface structure of the young rubber treeleaves. In Fig. 1D and F, the flexibility of the germ tubemorphology is seen and in Fig. 1E the formation of dark,contrasting material between fungus and plant is shown.Germ-tube diameter, germ-tube length, branching and for-mation of appressoria are strongly influenced by leafsurface constituents, which modify the infection process(Table 2; Blasquez and Owen, 1963; Hashim et al., 1978;Giesemann, 1986).

Penetration and initial infection stages

After penetration of the leaf surface, the hyphae colo-nize the underlying tissue by intercellular growth. Theyoften enter the tissue layers adjacent to the leaf vascularbundles and spread rapidly along the veins into theleaves (Fig. 1G, H; Giesemann, 1986). In this biotrophicphase, compatible combinations do not show cell death.However, in resistant clones, the cells in direct contact withthe penetration hyphae collapse. Hashim et al. (1978)ascribed this to a hypersensitive reaction and to pre-formedresistance factors (Blasquez and Owen, 1957; Figari, 1965),and proof was also given for induced defence compoundssuch as scopoletin (Tan and Low, 1975; Giesemann et al.,1986; Garcıa et al., 1995b). This early detection processof the fungal presence in the attacked tissue of resistantplants leading to a hypersensitive response is a typicaldefensive reaction (Breton et al., 1997). This reaction is


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FI G. 1. Anatomical and morphological aspects of M. ulei attack. (A) Heavyly infected leaflets of H. brasiliensis with greyish conidiospore layers onupper and lower leaf surfaces. (B) Dried conidiospores on lower leaf surface with reticulate surface structure. (C) Germinating conidiospore, section.(D) Germ tube adjacent to the wax layers on the reticulate surface structures of epidermal cells. The dark grey contrasted germtube is very flexibleand polymorphic. (E) Section through a germ tube that is fixed to epidermal cells by a slimy and sticky compound, which is set free from the conidiosporesurface after intensive washing. (F) Germinating conidiospores forming penetration structures: a penetration hyphae is entering the leaf at the junction ofthree adjacent cells (middle). (G) Blue-stained hyphae are spreading through the leaf tissue, often parallel to the vessels (24–36 h post-infection).(H) Blue-stained hyphae; branching hyphae growing back to the surface form dark-blue points (60–72 h post-infection). (I) Conidiospores break

through the lower leaf surface. (J) Black pigmented globose structures form ring-like stromata.


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regarded as an indicator for complete or vertical resistance,but this concept is applicable only to mature leaves ofH. brasiliensis and occurs with most genotypes of the pre-viously uninvestigated host species Hevea pauciflora(Junqueira et al., 1988). At the biochemical level of hostreactions, a hypersensitive response is often associatedwith well-described defence reactions such as formationof reactive oxygen-type compounds (Garcıa et al., 1999),deposition of autofluorescent compounds in the cell wall(Mevenkamp, 1992), synthesis of callose, occurrence ofscopoletin as phytoalexin (Giesemann et al., 1986;Garcıa et al., 1995b), and finally cell death in a restrictedarea surrounding the penetrating hyphae. Detailed andquantified descriptions have been given by Garcıa et al.(1999).

Fast hypersensitive response reactions typically lead totiny necrotic lesions or to yellow microlesions in controlled

inoculation experiments (Fig. 2) and they have also beenreported as field symptoms associated with both youngtissues and with mature rubber tree leaves. Thus, matureleaves express the well-described stage-specific resistance.All adult leaves (Blasquez and Owen, 1963; Junqueiraet al., 1988), irrespective of the clonal origin, are highlyresistant. This transient and stage-specific enhancement ofbiochemical resistance patterns needs to be considered inany new strategy of plant selection for broad-based resist-ance against M. ulei.

In the early phase of leaf penetration and colonization,cyanide liberation occurs as a consequence of cell break-down (Lieberei, 1986). Cyanide liberation is due to thecontact of vacuolar cyanogenic precursors with an apoplas-tic b-glucosidase. HCN, depending on the velocity of liber-ation and on precursor concentration, can be inhibitory toplant defence reactions or simply diluted out.

FI G. 2. Evaluation scheme of M. ulei lesions. The classification of lesions follows the description of Junqueira (1985). On leaf section ‘0’ only very tinynecrotic spots become visible. The lesions develop within 3–4 d after infection and incubation in a humid chamber under controlled conditions.

TABLE 2. Germination of M. ulei conidiospores on leaves of selected rubber tree clones: influence of host–pathogencombination in the pre-germination phase. On resistant-reacting leaves the germ tubes are shorter than on susceptible leaves,the germ tube diameter is bigger, and the amount of appresion formed is higher on resistant leaf surfaces. On susceptible

leaves, germ tubes are found to form branches

Control HS R/S R

H2O GT 1 RRIM 600 IAN 573 Fx 25 F 4542 PuA 7 F 2261

Germination (% in 24 h) 96 95.5 95 95.8 76 95.5 95.8 95.5Germ tube length (mm in 40 h) 124 114 127 56 62 70 66 73Germ tube width (mm in 40 h) 4.2 3.6 3.4 6.0 6.0 5.8 6.1 6.2Appressoria formed (% in 40 h) 5.2 5.8 5.5 19 17.5 14.5 22.2 20Branched germ tubes (%) 22 n.t 12 n.t n.t 8 n.t

Data taken from Giesemann (1986), n.t: not tested.


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Leaf colonization, intercellular hyphal growthand ramification

In susceptible host–pathogen combinations, the intercel-lular spread of the fungal hyphae takes place along the leafvein (Fig. 1G) and freely into the apoplastic regions of thetissue between the veins (Fig. 1H; Giesemann, 1986). Thisphase ends after 3–5 d with a remarkable morphogeneticchange in hyphal growth: the hyphae then form sidehyphae, which grow perpendicular to the original hyphae.This new type of hyphae can be seen as coloured pointsadjacent to the leaf-colonizing hyphae (Fig. 1H). The exter-nal or internal factors that regulate these morphogeneticchanges remain to be studied. These factors appear to belinked to the leaf maturation process. In the intercellularspaces around the growing hyphae, lignin-like material isdeposited that can lead to reduction of the lesion size.

Formation of conidiophores

The branching hyphae are directed towards the lower epi-dermis and within 24 h after formation they break throughthe lower leaf surface and form conidiophores and conidia(Garcıa et al., 1995a). Small lesions that can fuse laterallyto form ring-like structures often lead to large lesions(Fig. 2). The fact that the lesion formation patterns occurin defined types and in a reproducible manner indicatesthat intercellular growth and ramification of the hyphaeunderlies a plant genotype–fungal race interdependence(Junqueira et al., 1988).

Stroma production and ascospore formation

Stromatic structures with pseudothecia and ascosporesare formed on infected leaves with low infection incidence.Leaves with low levels of infection remain on the trees,whereas heavily infected leaves fall from the tree after con-idiospore formation and degrade on the ground. Whenless-infected leaves remain in the crown 2 months ormore after infection, stromata on the upper leaf side areformed in small globose structures that may fuse to formrings (Fig. 1J). In these structures, the sexual stages offungal development are completed with formation of ascos-pores. In many plant–fungus combinations, the formationof stromata does not occur. Junqueira et al. (1990) reportedthat conidial formation in lesions is dependent on theleaf age when infection takes place. The older the develop-ing leaves, the smaller the number of spores formed.Subsequently, formation of stromata is delayed.

Description of the plant–pathogen interaction betweenHevea species and SALB and the occurrence ofhost-specific reactions to various fungal pathogen racesestablishes that the large set of biochemical defenceevents known to occur in many other plants also occur inrubber trees and, furthermore, knowledge of these stepsunderscores the fact that a genetic precondition for bio-chemical defence is present in all rubber tree genotypes(Hashim et al., 1978, 1980; Mevenkamp, 1992; Garcıaet al., 1995a, b). Initiation and function of host defence pro-cesses depends on plant/pathogen signalling between host

genotypes and fungal isolates. These processes are linkedto successful and rapid detection of signals and to activationof the complete biochemical signalling chain for both ver-tical and complete resistance systems. In addition to thisgeneral defence based on major resistance genes, manyother variables and patterns in the formation of lesionsize and of spore formation are expressed (see Figs 2and 3). Some host plant genotypes are susceptible to allfungal isolates that have been examined. These variationsin expression of partial or horizontal resistance must bebased on genetic factors, but can be modified by transientfactors on the physiological level of plant hosts (Liebereiet al., 1989).

PHYSIOLOGICAL LEAF PROPERTIES

Rubber tree leaves are formed in a flush growth pattern.Directly after bud burst, the leaves are thin, have a high res-piration rate, no net photosynthesis (Table 3; Lieberei,1984; Lieberei et al., 1996) and are devoid of any resistanceagainst the virulent isolates of M. ulei. In the course ofmaturation, rubber tree leaves change from susceptible tocompletely resistant (Blasquez and Owen, 1963; Holliday,1970; Chee, 1980). This maturation requires 12–20 dafter bud burst to occur; the maturation time is genotypedependent. This parameter is very important for diseasemanagement. Based on morphological criteria seen frombud burst until hardening of leaflets or leaves, Dijkman(1951) placed the developing leaves in four groups desig-nated A to D (Fig. 4). Blasquez and Owen (1963), Halleand Martin (1968) and Chee (1976) used leaf colour andthe angle formed between the petiole and stem to placethese leaves into groups, whereas Samsuddin et al. (1978)correlated the developmental groups with the angleformed between the leaflets and the petiole. In contrast,Lieberei (1984) used physiological data for the descriptionof leaf developmental stages. Vob (2001) used leaf lengthincrease (Fig. 4), and Lieberei (1984) chlorophyll per leafarea, the chl a : b ratio, the fresh weight per leaf area, andselected data on gas exchange to describe the sink status(Table 3).

Based on these physiological data, leaf stages A, B and Care completely sink leaves with no positive net energybalance (Lieberei et al., 1996). Leaf size increase isalmost exponential in the early stages of leaf development.In this phase of rapid cell wall growth and cell enlargement,cell wall hardening processes are retarded; leaves in stagesA, B and C are almost free of lignin (Fig. 5A–C; Vob, 2001).

The amount of UV-absorbing compounds in 1-mm leafsections were analysed with a UV-microspectrophotometer(UMSP 80, Zeiss; Fig. 5). This microspectrophotometricanalysis permitted us to prove the occurrence of phenoliccompounds in the middle lamellae and primary cell walls.Leaves at stages B and C contain some aromatic com-pounds, but do not contain lignin. UV-photographs of theleaf sections, taken with UV 280 nm light, show that indevelopmental stages B and C only lignification in the trac-heids of conductive vessels occurs. Other cell walls aredevoid of lignin (Vob, 2001). The typical UV absorptionpattern of lignin with three peaks near 280 nm (see


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Fig. 5C) can only be detected in leaf structures of stage D.This presence of lignin coincides with the onset of leafhardening in early stage D.

Leaves of developmental stage D possess the physiologi-cal and structural parameters of mature leaves (Table 3,Fig. 5C). These leaves are completely resistant to M. ulei.The penetrating hyphae of all strains and isolates ofM. ulei tested are stopped by hypersensitive responses

(Fig. 3: necrosis as described by Blasquez and Owen1957, 1963).

With young leaves (stage B), fungal infection leads toincomplete defence reactions. In the course of these reac-tions, the soluble coumarin scopoletin is formed (Tan andLow, 1975; Giesemann et al., 1986; Garcıa et al., 1999).Scopoletin is a fungitoxic compound, but the concentrationsreached in Hevea leaf tissue are not very high. According to

FI G. 3. Biochemical resistance reactions in rubber tree leaves. (A) Formation of lignin in cell walls around lesions of intermediately resistant leavesinfected by M. ulei. There is a progressing impregnation of the cell walls with lignin (Vance et al., 1980). (B) Resistance reactions are accompaniedby the formation of wallbound insoluble phenolics, which become visible after treatment of leaves with Toluidin blue (Feder and O’Brien, 1968).(C) Callose formation around localized lesions (triangular zone in centre of image), detected with Aniline blue (Eschrich and Currier, 1964).(D) Necrotic spot in a hypersensitively reacting leaf adjacent to mycelium (myc); lep ¼ lower epidermis. (E) View of an infected leaf surface underUV-irradiation, red chlorophyll fluorescence in the background and faint blue fluorescence of scopoletin (scop); inoc ¼ inoculation site.(F) Scopoletin in a reticulate distribution under UV irradiation. The blue fluorescing field surrounds black germinating spores of Periconia manihoticola;leaf incubation 24 h under humid conditions in the dark in Petri dishes. Scopolin is formed when the leaves are left attached to the plant and are covered by

black plastic bags. The fluorescent compound is located in the vacuoles.


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Garcıa et al. (1999), the amount of scopoletin is not directlycorrelated to resistance.

DYNAMICS OF LEAF RESISTANCE:PERSPECTIVES OF LEAF FLUSH GROWTH

IN PLANT DEVELOPMENT

Rubber tree leaves from susceptible Hevea trees are highlysusceptible at leaf stage A, but become completely resistantat leaf stage D. The susceptibility of leaves of all Hevea

clones to infection with M. ulei generally declines withleaf maturation. This conversion of leaves is a transientand progressive process. The older the leaves are at thetime of inoculation, the more distinct is the defence reaction(Junqueira et al., 1990). Within the pathogen isolates,groups have been identified that vary with respect to infec-tion intensity and with respect to their host range (Junqueiraet al., 1988; Fig. 2). The variation of resistance of Heveaclones at different stages of leaf development is presentedin Figs 6 and 7. Leaves inoculated with virulent isolates

FI G. 4. Relationship between exponential leaf growth, transition phase from completely susceptible leaf stage to completely resistant leaf stage, and theshort physiological step from sink to source leaves. This qualitative comparison attempts to visualize the different developmental processes that turn asusceptible rubber tree leaf into a completely age-resistant organ. In the hatched area the resistance factors interact in various ways and do not allow

the correlation of the QTLs developed with the leaf properties in transition.

TABLE 3. Quantitative characteristics of developmental leaf stages of rubber trees

Leaf stage

A B C D

Chlorophyll (mg cm22 leaf area) 26 33–38 25–33 43–51Chl a/b ratio 1.2–1.6 1.8–2.2 2.2–2.9 3.3–3.6Photosynthetic CO2 fixation (mmol CO2 m22 s21) 20.72 20.24 5.1–11.6Photosynthetic CO2 fixation per unit chlorophyll (mmol CO2 mg21 Chl a s21) 16.1 15.3 12.0Respiration (mmol CO2 mg21 Chl a s21) 25.03 24.48 22.01Fresh weight (mg cm22) 19 16 9 11Dry weight (% of fresh weight) 18.9 19.6 40.7Leaf soluble protein (mg cm22) 1150 865 365 740HCN-p (mmol CN g21 fresh weight) 20 30 37 20


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FI G. 5. UV photographs and UMSP 80 scans of rubber tree leaves at different developmental stages. Lignin formation was analysed with an ultraviolet(UV) spectrometric device (UMSP 80, Zeiss). Semi-thin sections of acetone-extracted leaves of 1 mm were mounted on quartzglass trays. Spectra weretaken in the range of 250–280 nm. Leaves of stages B2, C and D were tested. 1, Middle lamellae and added material; 2, contact layer between threeadjacent cells 3; secondary cell wall. The typical absorption spectra for lignin with three combined absorption peaks between 275 nm and 280 nm

were detectable in leaf stage D, not before (Vob, 2001).


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at an early leaf age generally form spore-producing lesionsa short time after inoculation (Fig. 6). In addition to theshort time of the generation period, these lesions are largein diameter. The older the leaves are at the time of inocu-lation, the smaller in diameter the lesions become. Theexpressed clonal characteristics of susceptibility areanother important factor of disease variability. In cloneIAN 717 (Fig. 7), the young leaves produce spores 5 dafter inoculation and the lesions reach a size between2–4 mm in diameter. In clone IAN 6158, spore-producinglesions are less than 2 mm when inoculation takes place10 d after bud burst. When inoculation is carried out 12 dafter bud burst, small necrotic lesions do occur, but nospores are formed in these lesions (Fig. 6). This stage ofleaf maturation is characterized by a limited susceptibilityto penetration and early fungal spread in the leaf tissue,but later resistance factors such as lignification inhibit thelater developmental phases of the pathogen.

The susceptible leaf phases vary considerably with theclones studied (Fig. 7). Clones Fx 7 3925, IAN 873, andIAN 717 are susceptible until a leaf age of 16 d, whereasclones such as IAN 8 6158 or CNS-AM 79 07 becomeresistant with 10 d. In addition to the clone-specific,leaf-age resistance there is also variation in pathogen devel-opment on the clones due to variation in maturation time. Inleaves with a long leaf-maturation phase, fungal develop-ment is short, i.e. the fungal generation period, defined asthe time from inoculation until the formation of newspores (Fig. 7), is shorter than on leaves with short leaf-maturation times. This relation of fungal development toleaf maturation is very important if one considers epidemio-logical aspects. If the first leaf of a flushing rubber treecanopy is infected and produces spores in the shortesttime possible in clone Fx 3925, then by direct reinfectionof young leaves at least three fungal generations will be pro-duced, whereas in clones such as F 4542 or CNS AM 7907the production of spores in infected leaves almost coincideswith the onset of leaf-age resistance in the tree crown.Under these conditions reinfection by the newly producedspores in the canopy cannot occur.

FI G. 6. Fungal generation period and lesion diameter on leaves inoculatedat different days during the leaf flushing period. Leaves of selected cloneswere inoculated with M. ulei isolates highly virulent to the respectiveHevea clones. The ‘leaf age’ corresponds to the day of inoculation afterbud burst, the generation period is given in days from inoculation untilspore formation of 50 % of the lesions, and the diameter of lesions aregiven in mm. The data for the figure have been taken from Junqueira

et al. (1990).

FI G. 7. Fungal generation period in relation to leaf susceptibility period.


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The variation of lesion size on leaves at various stages ofmaturation represents a significant observation. The growthof the fungus through the host-leaf apoplastic space isreduced with increasing age of the leaf tissue. In a structuralstudy of infection-induced defence reactions of rubberleaves against M. ulei, Garcıa et al. (1995a) reported scopo-letin formation and accumulation of lignin-like substancesaround the infection site. These authors assumed that ligni-fication may stop fungal development. Lignification is anenergy-dependent process that is dependent on the avail-ability of precursors and reductive equivalents. Under theassumption that only source leaves are able to carry outdefensive reactions, it becomes important to conduct thesestudies under appropriate light conditions and with leavesattached to the mother plants. In the past many experimentshave been carried out by incubation of detached leaves inhumid chambers in the dark (e.g. Hashim et al., 1980;Lieberei, 1986; Garcıa et al., 1995b; Breton et al., 1997).

PLANT CYANOGENESIS: LEAF INHERENTCONSTITUENTS WITH HIGH INTERACTION

POTENTIAL WITH DEFENCE REACTIONS

In addition to the varying activities of phenolic metabolismand lignin formation, Hevea leaves are characterized by lib-eration of HCN upon infection and tissue injury. The rubbertree belongs to a large group of more than 3 000 species thatare cyanogenic, i.e. they release cyanide on damage to theplant. These plants accumulate cyanogenic precursors intheir vacuoles. Upon tissue injury the precursors, mostlyb-glycosides, are set free from the vacuoles and aremixed with b-glycosidases. Generally, these b-glycosidasesare stored separately from the cyanogenic precursors,mostly in the plant cell walls as active apoplastic constitu-ents (Selmar et al., 1988). The pre-formed enzymes cleavethe liberated b-glycosides and give rise to a sugar moietyand an aglycone. The aglycone decays and forms HCNand a corresponding carbonyl compound (Seigler, 1998).The time course of HCN liberation from the aglyconedepends on the leaf tissue, pH and on the occurrence ofanother enzyme, an a–hydroxynitrile lyase, which acceler-ates HCN-liberation (Selmar et al., 1989).

The cyanogenic precursors of the rubber tree are the glu-cosides linamarin and lotaustralin (Lieberei et al., 1985). Inthe course of leaf infection by M. ulei or otherleaf-attacking fungi, HCN is released from injured leaftissue. The liberation of HCN follows a reproducible timecourse. In the case of leaf infection by M. ulei, there is acharacteristic HCN peak in the early infection stage(Lieberei, 1986) about 24 h after penetration of the fungusinto the tissue.

Cyanogenesis is an obligate feature of rubber trees. Allliving tissues, including seeds, are strongly cyanogenicand contain both the accumulated cyanogenic precursorand the respective b-glucosidase.

The amount of cyanogenic precursor accumulated pertissue weight permits calculation of the cyanide liberationpotential of the tissue and is called the HCN-potential orHCN-p (Loyd and Gray, 1970). The rate of HCN liberation(HCN-c) from an attacked tissue depends on the type of

tissue injury and the activity of the b-glucosidases in thegiven tissue. In many cases this is also linked to the pre-sence and activity of an a-hydroxynitrile lyase (Selmaret al., 1989). Plants of Hevea brasiliensis as well as allother Hevea species studied are cyanogenic, although theyvary considerably in their HCN-p and in their HCN-c(Lieberei, 1984). There is a clear relationship of HCN-cof the rubber tree to the susceptibility to M. ulei. Thehigher the HCN-c, the higher the susceptibility.

In plants with low HCN-c and HCN-p values, the resist-ance reactions are not impaired by HCN. These plantsfollow a well-described host–pathogen reaction patternwith the complete spectrum of lesion formation (Lieberei,1988a, b). These plants have the classical race-specificresistance patterns. A more detailed examination of thisplant–pathogen interaction has been conducted with selectedpairs of rubber tree genotypes high in HCN-c and low inHCN-c and selected isolates of M. ulei, which were ident-ified by their host range (Lieberei et al., 1989).

When inoculated leaves were incubated in humidchambers, strongly cyanogenic leaves always developedsevere lesions, whereas weakly cyanogenic leaves developedthe defence patterns shown in Fig. 2. When HCN liberationwas experimentally minimized in the incubation systems byresorption of HCN in alkali or by passing a flow of humid airthrough the incubation system, the highly susceptible,strongly cyanogenic leaves reacted with formation ofsmaller lesions and with good resistance responses(Lieberei et al., 1989). These results proved that HCN libera-tion led to impairment of active defence in the leaves, andthat leaves of high HCN-p plants after experimental lower-ing of HCN in the tissue produce the entire set of defencereactions that can be expressed after signal perception.

HCN liberation is a pre-formed trait in plant tissue, whichmay provide a very good defence against herbivores (e.g.Tattersal et al., 2001; Ballhorn et al., 2005), but whichmay render strongly cyanogenic plants more susceptible tomicrobial pathogens (e.g. flax to flax wilt, Trione, 1960).The theoretical background of this controversial reactionneeds to be considered in greater detail as a complexphenomenon in quantitative ecology and evolution. For therubber tree, this phenomenon must be taken into consider-ation for practical breeding and selection work. The fungalpathogen M. ulei is tolerant to HCN, at least in its vegetativephase (Lieberei et al., 1983). In addition, the fungus pro-duces a soluble extracellular b-glycosidase that has remark-ably high affinity to the hosts’ cyanogenic compoundlinamarin (Lieberei, 2006). The quantitative potential inthe leaf material varies with leaf age. The youngest and gen-erally most susceptible leaves have pronounced HCN-p anda high activity of b-glucosidase whereas, during maturation,the leaves (stage D) often reveal lower values (Table 3).

MOLECULAR ANALYSIS OF GENETICDIVERSITY IN RUBBER TREES

In 1981, a germplasm collection excursion to the geo-graphical area of the primary rubber tree germplasm inthe Amazon was conducted by an international group,formed under the auspices of the International Rubber


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Research and Development Board. The resulting IRRDBcollection was the first systematic collection of Heveagermplasm in more than 100 years since the rubber seedcollection of Wickham in the Amazon basin. This newrubber germplasm collection was carried out in order tobroaden the genetic basis for worldwide rubber tree cultiva-tion and domestication. Studies of the genetic and molecu-lar diversity of the selected rubber tree germplasmsubsequently began within the Biotrop program fromCIRAD, France. Genome organization and molecular diver-sity were analysed, using 14 isozyme loci, cytoplasmic andnuclear RFLPs and, later, 17 microsatellite loci (Besseet al., 1994; Le Guen et al., 2003). The germplasm acces-sions could be separated into six genetic groups, whichencompass populations of defined geographic origins, spreadover the entire Amazon Basin and the western Andeanregion (1), the eastern region near the Tapajos river (2),the federal state regions of Acre (3,4), Rondonia (5), andMato Grosso (6) (Besse et al., 1994).

According to detailed studies by Seguin et al. (1996) andLespinasse et al. (2000b), Hevea brasiliensis has a diploidgenomic organization with rare duplicated loci. Thesestudies led to identification of 18 basic linkage groups ina rubber genome of 2150 cM total map length. Using the195 progenies of the population derived from the crossingof PB 260 � Fx 3899 and their response to six isolatedstrains of M. ulei, eight quantitative trait loci (QTL) withrespect to resistance were identified on seven independentlinkage groups.

Le Guen et al. (2003) and Lespinasse et al. (2000b), onthe basis of their molecular data, prepared the mapping ofgenes conferring field resistance to South American leafblight. For the first time, it was shown that both factorsfor partial resistance and for complete resistance were quan-titatively expressed in the progeny and could be correlatedwith five loci. The molecular approach to this plant–pathogen combination has greatly enhanced the possibilitiesof proceeding with marker-assisted breeding and selectionin a perennial tropical plant.

Nevertheless, some important questions remained to beanswered. The authors debated why the defence mechan-isms controlled by a locus termed M 13-lbn differedstrongly in behaviour between the field test and thatwithin an inoculation chamber: the efficiency was bestunder field conditions, but weak or ineffective under con-trolled conditions. In susceptible progeny, the resistanceconferred by M 13-1bn was no longer detectable.

The same question arises for the QTLs that correspond tolinkage groups g15 and g12. These factors are observableunder controlled conditions but are not found in field experi-ments. For this reason, whether the lesion-diameter trait is auseful predictor for SALB resistance under field conditionsis debated no matter what the environment. The hypothesisof a ‘QTL with a major effect modulated by a few otherminor QTLs’ was strengthened by further analyses. Ingeneral, the state of development of genetic analysis inthis tropical perennial plant is very promising and pursuingthe next steps to marker-assisted breeding appears feasible.

In a parallel step, characterization of the variability of theascomycete M. ulei is also proceeding well. Eleven

polymorphic microsatellite markers for M. ulei have beencharacterized and applied to six isolates from Brazil andfour isolates from French Guiana.

Recent genetic studies of rubber tree resistance to M. uleiindicate that, in addition to the eight major resistance-conferring QTLs, there must be minor factors that modulatethe action of the major components or that interfere with thephenotypic expression of these components. The majorresistance factors, when active, result in total expressionof defensive reactions and lead to complete resistance, i.e.to control of fungal development in the leaf; this ultimatelymeans that no lesions or no spore-forming lesions occur(lesion types 0, 1 and 2, Fig.2). These lesion types dooccur and give proof for the occurrence of major resistancefactors.

But the conclusions drawn from the molecular studiesalso give clear evidence that in addition to the majorgenes for complete resistance, a set of minor componentsinterfere with the performance of the major resistancefactors.

From the results of previous studies on resistance physio-logy of rubber to M. ulei, the importance of various typicalbiochemical properties of the rubber tree that can activelyinterfere with resistance reactions on several levels isknown. These reactions occur when the major gene resist-ance is overcome by pathogen isolates with their respectivevirulence genes. In these combinations of virulent pathogenand susceptible host, the minor genes or the polyfactorialresistance factors are expressed. Their action is expressedby quantitative variation in lesion size and spore abun-dance. Some of these polyfactorial reactions have beendescribed with respect to scopoletin and lignin formationby Garcıa et al. (1999), but the studies have never beenevaluated from the aspect of transient changes of leaf physi-ology in the course of the infection process. In the samecontext, resistance impairment by the process of cyanogene-sis has never been regarded as interacting with the classicalresistance processes. So far, the impact of cyanogenesis hasnever been considered to be important for the outcome of adisease response, though the studies of Lieberei (1986,1988a) and Lieberei et al. (1989) clearly established thatstrongly cyanogenic Hevea genotypes have inefficient orno defence responses. Consequently, this raises the questionas to what extent the two properties ‘sink leaves’ and ‘cya-nogenesis’ interact with resistance such that they canexplain some of the irregularities identified in studies onbiochemical defence responses by Garcıa et al. (1999).

Leaf flush growth is a remarkable and special feature oftropical plants such as rubber trees (Halle and Martin,1968). Leaf stages A, B and C of rubber are metabolicsink leaves and their transition from a metabolic sink to ametabolic source organ is remarkably long. Some of theleaf parameters are summarized in Table 3, which isbased on a study by Lieberei (1984). The amount of chlor-ophyll per leaf area increases from leaf stage A, with26 mg cm22, to leaf stage D with 47 mg cm22, and theChl a/b ratio shifts from a low value to the typical valuefound in photosynthetically active plants of about 3. Leafstage C possesses a remarkably lower chlorophyll amountper area than either stage B or D. The fresh weight per


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area is lowest in stage C, as is the amount of soluble proteinper area. These data are a consequence of exponential leafgrowth. The leaf area from stage B to stage C is enlargedmainly by leaf water uptake and cell enlargement. Duringthis phase, the cell walls are very thin and flexible andcell wall hardening is minimal. Lignin is definitely absentfrom cell walls until stage D (Vob, 2001). On the otherhand, it is remarkable that the vacuolar content of cyano-genic precursors per fresh weight rises from stage A tostage D and the activity of the apoplastic b-glucosidase/linamarase also is very high (Table 3, and Lieberei et al.,1985). Cell wall changes and lignin formation obviouslybecome a predominant reaction with the onset of leafstage D. The ratio of dry weight to fresh weight doublesfrom stages B and C to D due to cell wall lignification,and this will also be the reason for the obligate restrictionof fungal spread in the wall region of stage D leaves.

The leaves are metabolic sink tissues and dependent onthe energy balance of the mother plant for a long time.Energy-dependent synthesis requires transport of assimilateinto the leaves to occur. Under these conditions, it seemsclear that resistance-screening experiments carried outwith detached leaves will result in different results fromthose with leaves attached to the plants. Processes such ascinnamic acid synthesis or changes in pool sizes of aminoacids, active synthesis of scopoletin, lignin and glycosideswill be retarded or even stopped in detached leaves due toexhaustion of energy-delivering compounds.

In tests with leaves attached to the plants under controlledconditions, pathogen development will perform differentlybecause these leaves receive their metabolic supply fromthe adjacent source organs. The production of scopoletin,which is produced as a response to fungal attack in rubbertree leaves (Breton et al., 1997; Garcıa et al., 1999), isalso dependent on the energy-delivering sources in the inoc-ulated leaves. The amount of scopoletin accumulated in theinoculation droplets, as followed by Garcıa et al. (1995b,1999) and by Breton et al. (1997) is the result of several reac-tions: the scopoletin formation as an induced response topathogens and to tissue injury, the elicitor treatment, andother forms of stress to the plant tissue. Scopoletin isexuded from the living cell into the apoplast and caneasily be detected under UV excitation as a blue fluorescentcompound. Because of its weak antimicrobial action, scopo-letin is regarded as a phytoalexin in Hevea species(Giesemann et al., 1986). In the cell wall, apoplastic peroxi-dases convert scopoletin in the presence of H2O2 to yellow/brown insoluble substances and the fluorescence is quenched(Breton et al., 1997; Chong et al., 1999). A second typicalreaction lowering the scopoletin content is the glycosylationof scopoletin to yield the glucoside scopolin. Scopolin isformed by the action of UDP-glucosyl transferase (UGT);the glucoside is located in the vacuole. Recently Langlois-Meurinne et al. (2005) reported on the stress- and pathogen-related expression of UGT in plant–pathogen interactionsand, in a general approach, Gachon et al. (2005) emphasizedthe importance of glycosylation for biosynthesis and storageof secondary compounds. In fact, in most plants that producescopoletin in response to pathogen attack (e.g. potato,Hughes and Swain, 1960; tobacco, Sequeira, 1969;

sunflower, Tal and Robeson, 1986), scopolin co-occurswith scopoletin. In rubber tree experiments, Garcıa et al.(1995b, 1999) and Breton et al. (1997) did not detect scopo-lin. In contrast, Korner (1994) found both scopoletin andscopolin after infection of H. brasiliensis leaves withspores of the weak Hevea pathogen, Periconia manihoticola.Korner (1994) infected rubber tree leaves (stage B) withspores of P. manihoticola and incubated them as detachedleaves in darkened Petri dishes on moistened filter paper at22 8C and as leaves attached to the plant, covered by ablack plastic bag that also contained a moistened filterpaper. After 2 d, small necrotic lesions about 1 mm in diam-eter surrounded by a yellow halo had developed under bothtreatments. When analysed under UV-light, both treatmentsproduced a blue fluorescence in the tissue. The fluorescencein detached leaves was found as a net-like distribution in theapoplast, whereas the fluorescence in the attached leaves wasfound to be concentrated in the vacuoles (Fig. 3). Aftermethanolic extraction, these two compounds could be veri-fied by TLC analysis as scopoletin in detached leaves andas scopolin with trace amounts of scopoletin in leavesattached to the plants. The general difference between theinoculations that produced scopoletin or scopolin was thefact that leaves attached to the plants appeared to receive suf-ficient assimilates from the mother plant to form the gluco-side, whereas the detached leaves, as isolated sink organs,used reserve compounds to produce limited amounts of sco-poletin, but did not possess enough reserve substances toprovide energy and substrates for carrying out UDP-glucose formation. In this context, the experimental use ofdetached leaves for resistance screenings must be re-evaluated. The amount of scopoletin produced in detachedleaf systems has also been demonstrated to be highly influ-enced by incubation conditions (Lieberei et al., 1989).When leaves of strongly cyanogenic genotypes ofH. brasiliensis are inoculated and incubated in closed Petridishes, they form large lesions and large amounts of HCN(38.6+ 4.4 mg HCN g21 d. wt) are liberated. The amountof scopoletin is small (0.48 mg Scop g21 d. wt).

When the incubation conditions are modified in such waythat the HCN liberated from the leaves is immediately takenout of the system by absorption in alkali or by passinghumid air through the incubation chamber, the lesions inthe highly cyanogenic leaves do not develop to largersizes but remain small, and the amount of scopoletinformed around these lesions is about five times higherthan in the strongly cyanogenic condition. Thus, highcyanide liberation during pathogen development in thehost tissue can lead to impairment of active defence, or inother words, the experiments indicate that strongly cyano-genic plants may contain all potential resistance factors inthe same manner as resistant plants but these resistancefactors are impaired by another plant factor, in this casecyanide liberation (Lieberei et al., 1989).

Effects of cyanogenesis: importance ofthreshold concentrations

HCN liberation in plants can impair active defence(rubber tree, Lieberei, 1984, 1986; Lieberei et al., 1989;


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flax, Ludtke and Hahn, 1953; Trione, 1960). This finding ishardly accepted by breeders, because it is counter-intuitiveto the usual understanding of evolutionary mechanisms. Itshould be impossible to inherit susceptibility, but bothqualitative and quantitative aspects of cyanogenesis areinherited.

A more in-depth look into cyanogenesis indicates that itis a phenomenon in the framework of polyfactorial evolu-tionary concepts. Cyanogenic plants can be generallydivided into obligate cyanogenic plants and facultative cya-nogenic species. In obligately cyanogenic plants, the genesfor biosynthesis of cyanogenic precursors and theirvacuolar storage and the genes for the synthesis of theb-glycosidases specific for each cyanogenic precursor areconstitutively expressed. There are many quantitative varia-tions in this phenotypic feature among the cyanogenicplants. Many genotypes contain small amounts of cyano-genic compounds in low micromolecular ranges per tissuedry weight, whereas other genotypes of strongly cyanogenicplants such as Phaseolus lunatus, Manihot esculenta, Heveabrasiliensis, Sorghum bicolor, Passiflora capsularis andmany others accumulate large amounts of cyanogenicprecursors and the tissue content varies from less than1 mmol g21 d. wt to several hundred mmol21 d. wt oftissue (e.g. Lieberei, 1988a for Hevea species; Busk andMøller, 2002, for Sorghum bicolor; Ballhorn et al., 2005,for Phaseolus lunatus; Amelot et al., 2006, for Passifloracapsularis).

With respect to plant–herbivore interactions, perennialwoody plants are highly interesting models for the studyof polyfactorial environmental pressure. During the lifespan of leaves ontogenetic changes in HCN-p and othermetabolic patterns occur. This leads to variation of the bio-chemical scenarios presented to herbivores and leaf-attacking microbes (e.g. Gleadow and Woodrow, 2000a, b,2002; Miller et al., 2006).

Cyanogenic potential HCN-p (Loyd and Gray, 1970) andthe capacity for HCN release per unit time (HCN-c) allowus to quantify cyanide liberation as a reaction to tissueinjury. In addition to genotypic variability for HCN-p andHCN-c, tissues often reveal strong variability at differentontogenetic stages. Generally, young leaf stages have higherHCN-p and HCN-c than older leaves (e.g. Gleadow andWoodrow, 2000a, b).

Once liberated, HCN acts as a highly mobile compo-nent in the leaf tissue. Within seconds, this compoundreaches the cells surrounding the locus of HCN-production.HCN is an inhibitor of many biochemical processes(Solomonson, 1981) and impairs active defence in cells sur-rounding the lesion (Lieberei, 1988a; Lieberei et al., 1989,1996).

NEW APPROACHES IN MARKER-ASSISTEDBREEDING: HOW TO COMBINE COMPLEX

PHYSIOLOGICAL PARAMETERS WITHMOLECULAR MARKERS

Identification of major components that contribute to QTLspermits identification of potentially resistant genotypes.The plants that contain these QTLs generally possess all

metabolic systems needed for active defence and, thus,these genotypes are highly interesting for further breeding.When characterized by QTLs for production of largeamounts of high quality latex, these plant materials are aprimary choice for breeding and selection. The most suit-able plants for further development out of this group arethose plants with a short ontogenetic transition from com-plete sink leaves to complete mature source leaves. It isnecessary to check how long and to what extent youngleaves are devoid of sufficient energy reserves for the pro-cessing of active, synthesis-dependent defence reactions.When significant formation of scopoletin and the onsetof lignification are necessary for limitation of fungal devel-opment, a complete and successful response depends onboth the presence of an active phenylalanine ammonialyase (PAL) and a sizeable pool of aromatic aminoacids, especially phenylalanine, for a successful resistanceresponse.

The resistance-impairing effects of cyanogenesis have tobe considered independently from the leaf developmentstage. Cyanide inhibits active defence by inhibition ofphotosynthetic CO2-fixation and by inhibition of a numberof other enzymes (Solomonson, 1981), among them thekey enzyme for cinnamic acid production, PAL. Hansonand Havir (1972) reported in detail on the inhibition kin-etics for PAL. Korner (1994) repeated these experimentsand evaluated the same inhibitory response for PALextracted from leaves of H. brasiliensis. This finding alsoexplains the report of Lieberei et al. (1989), who showedbetter resistance when cyanide concentrations were exper-imentally lowered in inoculated leaves. Furthermore, unpre-dictable test responses for clones GT1, IAN 873 and othergenotypes that are known to be strongly cyanogenic (Garcıaet al., 1999) can be explained in this manner. Cyanide inhi-bition may also explain the discrepancy with some plants ofgreater susceptibility under in vitro conditions in compari-son with field conditions (Lespinasse et al., 2000a).

In general, this new approach for making selections ofplant material for breeding studies can be simplified byavoiding use of strongly cyanogenic genotypes in experi-ments. Furthermore, only genotypes with short flushingperiods and fast leaf maturation patterns should be used.Additionally, plants should be included that have irregularleaf shedding instead of regular and complete change ofthe canopy. The most promising genotypes are those withgenes from H. benthamiana (Junqueira et al., 1990).

MOLECULAR METHODS CAN BE USEDTO LOWER PLANT CYANOGENESIS

Techniques for application of antisense technology toproduce lower cyanogenesis levels have been successfullyaccomplished with respect to cassava. Both the groups ofMøller (Kristensen et al., 2005) and Sayre (Siritungaet al., 2004) offer excellent molecular approaches to thisfield. Whenever weakly cyanogenic plants are selected, itshould be verified to what extent a certain threshold levelof cyanogenesis confers on a plant protection from pestsand pathogens. Pathogens and pests employ different


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strategies to respond to cyanogenesis and other plant defen-sive factors. Evolution is dynamic, and so are our problems.

ACKNOWLEDGEMENTS

The technical, linguistic and idealistic support of VolkerNolting, Dirk Selmar, Karin Puttfarken, David Seigler,Christoph Reisdorff, Daniel Kadow and Gerald Koch aregratefully acknowledged. Parts of these studies have beencarried out in close collaboration with many colleaguesand friends of the Brazilian Agricultural Research Organ-ization EMBRAPA in Central Amazonia, Manaus,especially Nilton T.V. Junqueira, Luadir Gasparotto andVincente H.F. Moraes. The research projects have beensubstantially supported by DFG Li 238/1 and /2.

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Bahia DB, Pinheira E, Gomes ARS, Valois ACC, Goncalves PS,Melho JRV, Pereira JP. 1985. Clones de Seringueira (Hevea sp.(HBK) Muell. Arg.): origem e ancestralidade. Ilheus, Brazil:CEPLAC, 428pp.

Ballhorn DJ, Lieberei R, Ganzhorn JU. 2005. Plant cyanogenesis ofPhaseolus lunatus and its relevance concerning herbivore plant inter-actions: the importance of quantitative data. Journal of ChemicalEcology 31: 1445–1473.

Belgrave WNC. 1922. Notes on the South American leaf disease ofrubber. Journal of the Board of Agriculture of British Guiana 15:132–138.

Besse P, Seguin M, Lebrun P, Chevallier M.H, Nicolas D. 1994. Geneticdiversity among wild and cultivated populations of Hevea brasiliensisassessed by nuclear RFLP analysis. Theoretical and Applied Genetics88: 199–207.

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South American Leaf Blight of the Rubber Tree (Hevea spp.): New

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