Cyanogenesis of Hevea brasiliensis during Infection with Microcyclus ulei1)

Eight Hevea species have been shown to be cyanogenic. They all liberated HCN following mechanical tissue injury. Infection of Hevea leaves with conidia of the plant pathogen Microcyclus ulei leads to a large reduction of hydrocyanic acid potential, while only small amounts of HCN are set free from the leaves into the atmosphere. HCN production by infected leaves follows a reproducible pattern with a maximum between 40 and 60 hours after infection. During the entire time of infection free HCN can be detected in the leaves. From leaves of susceptible clones high amounts of HCN are liberated whereas from resistant clones only very little HCN is released. In Hevea infections with M. ulei, cyanogenesis does not lead to defense of the fungal pathogen but impairment of the resistance reaction.     

A newly identified locus controls complete resistance to Microcyclus ulei in the Fx2784 rubber clone

Using cultivars which are genetically resistant to South American leaf blight (SALB) caused by the fungus Microcyclus ulei is the only way to plant rubber trees in disease-affected areas. Numerous field observations led to the hypothesis that the resistance of the cultivar Fx2784 to SALB is likely to be monogenic. In this study, we investigated this hypothesis by examining the distribution of the trait in a cross between the resistant cultivar and a susceptible one. The individuals resulting from this cross were planted in field trials in French Guiana and Brazil. The resistance of all the trees was assessed by field observations. Bulk segregant analysis (BSA) using microsatellite markers was performed in French Guiana to determine which markers were genetically linked to resistance, and the results were validated by field observations in Brazil. In both locations, a 1:1 segregation of the resistance trait was observed, thus reinforcing the monogenic hypothesis. BSA showed tight linkage between resistance and the microsatellite markers located in linkage group 2 in the Hevea genome and enabled to pinpoint the resistance locus. The location was confirmed by observations on the trees planted in Brazil. This result should facilitate the use of Fx2784 resistance in future breeding programs for SALB resistance. This is the third major locus conferring resistance to SALB identified in rubber tree (Hevea spp.). These three loci are genetically independent, a favorable situation for genetic improvement of SALB resistance.     

A Comparison of Abscission of Rubber (Hevea brasiliensis) Leaves Infected with Microcyclus ulei with Leaf Abscission Induced by Ethylene Treatment, Deblading and Senescence

Studies on the histology and on effects of growth substancesand phenols as well as changes in activities of pectinmethylesterase indicated that the mechanism of abscission of Hevealeaflets infected with Microcyclus ulei differed from the mechanismof abscission of debladed, ethylene treated and senescent leaves.An abscission layer which was formed during abscission of debladed,ethylene-treated and senescent leaves was absent during abscissionof heavily diseased leaves. The ratio of pectinmethyl esteraseactivities in tissues distal to the abscission zone to activitiesin tissues proximal to the zone decreased in debladed and ethylenetreated leaves but such decreases were not detected during abscissionof Hevea leaves infected with M. ulei. Hevea brasiliensis Muell. Arg., rubber, leaf abscission, Microcyclus ulei, ethylene, indol-3-ylacetic acid, kinetin     

Identification of QTLs involved in the resistance to South American leaf blight (Microcyclus ulei) in the rubber tree

South American leaf blight (SALB) is a disease of the rubber tree caused by the fungus Microcyclus ulei. Quantitative trait loci (QTLs) for resistance were mapped using 195 F₁ progeny individuals derived from the cross between a susceptible cultivated clone, PB260, and a resistant clone, RO38, derived from interspecific hybridization. The resistance level of the progeny individuals was evaluated in controlled conditions. The reaction type (RT) and the lesion diameter (LD) were measured on immature leaves after artificial inoculation of the fungus. Five different strains of the fungus were used, all highly sporulating on PB260. Among those, four did not sporulate and one sporulated partially on RO38. Both pseudo-testcross parental genetic maps and the consensus map were constructed. The search for QTLs was performed using the Kruskal-Wallis marker-by-marker test and the Interval-Mapping method for the three maps. Eight QTLs for resistance were identified on the RO38 map. Only one QTL was detected on the PB260 map. The analysis of the F₁ consensus map confirmed results obtained with the parental maps. A common QTL was detected for resistance to the five strains for both RT and LD. Two QTLs were common for complete resistance to four strains, for RT and LD respectively. Resistance determinism for complete and partial resistance, and perspectives for breeding for durable resistance to SALB are discussed. Received: 1 August 1999 / Accepted: 27 August 1999     

High-quality genome assembly of Pseudocercospora ulei the main threat to natural rubber trees

Pseudocercospora ulei is the causal agent of South American Leaf Blight (SALB), the main disease affecting Hevea brasiliensis rubber tree, a native species to the Amazon. Rubber tree is a major crop in South American countries and SALB disease control strategies would benefit from the availability of genomic resources for the fungal pathogen. Here, we assembled and annotated the P. ulei genome. Shotgun sequencing was performed using second and third generation sequencing technologies. We present the first P. ulei high-quality genome assembly, the largest among Mycosphaerellaceae, with 93.8 Mbp, comprising 215 scaffolds, an N50 of 2.8 Mbp and a BUSCO gene completeness of 97.5%. We identified 12,745 protein-coding gene models in the P. ulei genome with 756 genes encoding secreted proteins and 113 genes encoding effector candidates. Most of the genome (80%) is composed of repetitive elements dominated by retrotransposons of the Gypsy superfamily. P. ulei has the largest genome size among Mycosphaerellaceae, with the highest TE content. In conclusion, we have established essential genomic resources for a wide range of studies on P. ulei and related species.     

Dispersal of conidia of Dothidella ulei from Hevea brasiliensis

South American leaf blight caused by Dothidella ulei occurs only in tropical America, on both indigenous and cultivated Hevea spp. The conidium (Fusicladium macrosporum) is a 1-septate, dry, air-borne spore about 40 × 7 μ, occurring on the abaxial surface of dry leaves in dense, powdery, olive-green masses, and with one or both cells collapsed. The conidia adhere to the surface of water droplets, becoming turgid, and are disseminated in splash droplets. A Hirst volumetric trap, placed within a prepared source in north-west Trinidad, showed a diurnal periodicity of conidial production, with a maximum at 10.00 h and minima at night or in the early morning. On rainless days there was also a minor peak at 20.00 h. Transient increases occurred after rain, most of which fell around noon. On wet days almost equal numbers of conidia were dispersed between 10.00 and 12.00 h. Large increases occurred in 87% of all rain showers between 09.00 and 13.00 h. After 13.00 h fewer rain showers caused such increases; the lowest (36%) was between 21.00 and 01.00 h. Twice as many were trapped on sunny days (> 9 h sun) at 09.00 h when there was full sunshine, compared with overcast days (< 5 h sun). A more clearly defined morning maximum occurred on relatively windy days, compared with calmer ones. Conidial sporulation became very low, or ceased, where rain fell below a mean of 3–4 mm per day for at least 20 days. Abundant sporulation occurred with a daily rainfall about twice this amount. The results support the belief that if Dothidella ulei appeared in Malaysia its spread would be rapid and its effects damaging.     

A rubber tree��s durable resistance to Microcyclus ulei is conferred by a qualitative gene and a major quantitative resistance factor

The components of genetic resistance from the Hevea brasiliensis cultivar MDF 180 against South American Leaf Blight (SALB) caused by Microcyclus ulei were investigated by QTL mapping. MDF 180 has already been described as a cultivar with a high level of partial and long-lasting resistance. The resistance of progeny individuals from a cross between a susceptible cultivar and MDF 180 was assessed both under controlled conditions of inoculation by three M. ulei isolates and under natural infection in a field trial. Genetic maps of the two parents of this progeny were mainly established based on microsatellites and AFLP markers. No resistance QTL were found in the susceptible parent. In the resistant parent, we identified a qualitative gene responsible for the resistance against isolates from French Guiana and a major quantitative resistance factor determining the resistance against isolates from the state of Bahia (Brazil). The qualitative resistance gene was denominated M15md and was located in the linkage group g15. Four minor resistance QTLs were also identified, two of which showed an epistatic interaction with M15md. The durability of the resistance of MDF 180 is discussed in light of these data.     

Guayule and Russian Dandelion as Alternative Sources of Natural Rubber

ABSTRACT

Natural rubber, obtained almost exclusively from the Para rubber tree (Hevea brasiliensis), is a unique biopolymer of strategic importance that, in many of its most significant applications, cannot be replaced by synthetic rubber alternatives. Several pressing motives lead to the search for alternative sources of natural rubber. These include increased evidence of allergenic reactions to Hevea rubber, the danger that the fungal pathogen Microcyclus ulei, causative agent of South American Leaf Blight (SALB), might spread to Southeast Asia, which would severely disrupt rubber production, potential shortages of supply due to increasing demand and changes in land use, and a general trend towards the replacement of petroleum-derived chemicals with renewables. Two plant species have received considerable attention as potential alternative sources of natural rubber: the Mexican shrub Guayule (Parthenium argentatum Gray) and the Russian dandelion (Taraxacum koksaghyz). This review will summarize the current production methods and applications of natural rubber (dry rubber and latex), the threats to the production of natural rubber from the rubber tree, and describe the current knowledge of the production of natural rubber from guayule and Russian dandelion.     

Some factors influencing spore germination and penetration of Alternaria longipes

The ranges over which the germination of conidia of Alternaria longipes was > 50% were 10–35 °C on agar and 15–30 °C on tobacco leaf disks. Germination was optimal at 22.5 °C; so was germ-tube growth, reaching c. 300 and 102 μm on agar and leaf disks respectively after 12 h. On average, 27% more conidia germinated and germ-tubes were 62% longer on disks from leaves washed for 5 min under running water than on disks from unwashed leaves. At controlled saturation deficits germination after 8 h at 1.1 and 2.3 mb was 42.3 and 9.3% respectively and the rate of germ-tube growth was < 0.8 μm/h, compared with 94.4% and 8.3 μm/h in standing water. These results, together with some field data, suggests that germination in the field is largely restricted to periods when free moisture is present on leaves. In Malawi, leaf temperatures and the duration of dew at night were adequate to allow germination and penetration in the absence of rain. Pollen, when applied with the inoculum, had little effect on the number of germinated conidia, but caused a c. tenfold increase in the number of successful penetrations.     

Biosynthesis of scopoletin in Hevea brasiliensis leaves inoculated with Phytophthora palmivora

Inoculation of resistant (R) and susceptible (S) Hevea brasiliensisleaves with Phytophthora palmivorainduced foliar necrosis and biosynthesis of scopoletin (Scp), considered as a Heveaphytoalexin. The degree of resistance of four clones, as classified by the necrotic lesions, was related to the rapidity and intensity of Scp production. The resistant BPM-24, and marked partially resistant clone PB-235, displayed an early secretion of scopoletin that intensified and lasted longer than the weak partially resistant RRIT251, and susceptible clone RRIM600. The lesion size and amount of Scp after infection were positively correlated to the concentration of spores applied to Hevealeaves. In addition, in leaflets inoculated with high spore concentration, Scp reached the highest level earlier than those with low spore concentration. A fungitoxic effect of Scp on mycelium growth was shown in bioassays; the I₅₀ value tested on Phytophthora palmivora was relatively the same as on Phytophthora botryosa, but much lower than those found on other leaf pathogens of rubber tree, Corynespora cassiicolaand Colletotrichum gloeosporioides. The lesion size and level of Scp upon spore inoculation may be appropriate for classifying Heveaclones according to their R/S with respect to P. palmivora     

Chemical treatment of soil to prevent transmission of tobacco rattle virus to potatoes by Trichodorus spp

Alternaria longipes (Ell. &Ev.) Mason survived on autoclaved maize stems for 6 months without losing its pathogenicity, but rapidly lost viability on non-autoclaved stems and could not be re-isolated 4 months after inoculation. In laboratory tests it infected both living and dead maize leaves. Some Alternaria isolates from non-solanaceous hosts infected tobacco leaves kept at high humidities for 10 days after inoculation, but not when this incubation period was reduced to 48 h. In the field, perennation on plants other than tobacco is unlikely to be important as a source of inoculum. Pathogenicity of Alternaria isolates was maintained from one season to the next when stored as conidia in sterile soil, or as dried, infected tobacco leaves; some isolates maintained on agar slopes under oil were still pathogenic after 5 years. Alternaria conidia collected from the surface of tobacco seedlings, and isolates from apparently healthy seedling leaves were pathogenic to mature tobacco. In the field conidia were detected on tobacco leaves soon after these emerged, and epiphytic colonies were occasionally found well in advance of symptoms. Many latent infections were also detected up to 5 weeks in advance of symptoms. Visual development of latent infections closely coincided with the end of leaf expansion.     

In vitro spore germination and infection of cultivars of Rubus and Rosa by downy mildews from both hosts

The cardinal temperatures for in vitro germination of conidia of imported and indigenous isolates of downy mildew from hosts in the genera Rubus and Rosa were similar. A high percentage of conidia germinated above 2°C and germination remained between 80% and 90% up to 15°C or 20°C, depending on the isolate. The highest incidence of disease on leaf disks of Tummelberry (blackberry × red raspberry) inoculated with an isolate of Peronospora rubi occurred at c. 15°C, with infection over a range from 2°C to 28°C. Tests on leaf disks in vitro, and leaflets of primocane and lateral shoots in plastic tunnels, with three hybrid berry (blackberry x red raspberry), six blackberry and nine red raspberry cultivars showed the hybrid berries to be most susceptible. In a plastic tunnel infected drupelets of red raspberry fruits developed more slowly and failed to ripen evenly compared with uninfected drupelets. Similar malformation of infected fruits occurred in a plantation of Tummelberry. An isolate of P. rubi attacked severely both Tummelberry and rose cv. Can Can. Fluorescence microscopy after staining with aniline blue showed that leaf disks of Tummelberry were extensively colonised by intercellular mycelium of P. sparsa isolated from rose, even though sporulation was sparse or absent. This supports the view that P. rubi and P. sparsa may be conspecific. Oospores of P. rubi were found routinely within leaf disks of Rubus cultivars inoculated in vitro and once in naturally infected leaflets of Tummelberry.     

The roles of ascospores and conidia of Pyrenopeziza brassicae in light leaf spot epidemics on winter oilseed rape (Brassica napus) in the UK

Ascospores of Pyrenopeziza brassicae were produced in apothecia (cup‐shaped ascomata) on oilseed rape debris. The conidia, which were morphologically identical to the ascospores, were produced in acervular conidiomata was greater than for lesions caused by ascospores. In June 2000, on the ground under a crop with light on the surface of living oilseed rape tissues. Ascospores were more infective than conidia on oilseed rape leaves. The proportion of lesions caused by conidia located on leaf veins leaf spot, numbers of petioles with apothecia decreased with increasing distance into the crop from the edge of pathways. Air‐borne ascospores of P. brassicae were first collected above debris of oilseed rape affected with light leaf spot on 5 October 1998 and 18 September 1999,12 or 23 days, respectively, after the debris had been exposed outdoors. P. brassicae conidia were first observed on leaves of winter oilseed rape on 6 January 1999 and 15 February 2000, respectively, after plots had been inoculated with debris in November 1998 and October 1999. In 1991/92, numbers of ascospores above a naturally infected crop were small from January to April and increased in June and July. P. brassicae conidia were first observed in February and the percentage plants with leaves, stems or pods with light leaf spot increased greatly in May and June. In 1992/93, in a crop inoculated with debris, numbers of airborne ascospores were small from October to January and increased from April to June. P. brassicae conidia were first observed on leaves in late November and light leaf spot was seen on stems and pods in March and June 1993, respectively.     

Dispersal of Rhynchosporium secalis conidia from infected barley leaves or straw by simulated rain

Simulated rain (mean drop diameter c. 1 or 3 mm) was allowed to fall for 10 – 15 min on to barley leaves or straw infected by Rhynchosporium secalis (leaf blotch). The leaves were supported on a mesh through which run-off water drained and the straw was supported on a rigid surface on which run-off water collected. The numbers of R. secalis conidia and spore-carrying splash droplets collected by horizontal samplers (microscope slides and pieces of photographic film) decreased rapidly with increasing distance from and increasing height above the sources, with half-distances of 2 – 10 cm. Less than 10% of the spores or droplets reached heights of more than 30 cm. Incident drops 3 mm in diameter produced more spore-carrying droplets and dispersed more conidia than did 1 mm drops. The size category of splash droplets with the greatest proportion of the spore-carrying droplets dispersed by 3 mm drops was 200 – 400 μm, whether the source was infected barley leaves or barley straw. For leaves or straw the greatest proportions of spores were carried in droplets > 1000 μm in diameter. The mean diameter of spore-carrying droplets (478 μm) dispersed from free-draining leaves was less than that of droplets from straw plus run-off water (563 μm). However, the leaf source had more spores cm^-2 and the mean number of spores per droplet was greater (113 as opposed to 6·8) than for the straw source.     

Effects of population density on alienicolae of Aphis fabae Scop.: The expression of migratory urge among alatae in the field

Inspection of naturally or artificially infected Hevea roots showed that Forms lignosus can penetrate undamaged roots directly, but does so more readily through wounds or natural openings like lenticels, or through the bases of lateral roots and bark scales. Therefore, Pomes-infected trees should be identified by leaf symptoms rather than by uncovering and inspecting roots, as this generally leads to root injury, which facilitates fungal penetration. Initial fungal entry into host tissue appears to be by mechanical pressure alone, but deeper penetration is through the action of extracellular enzymes. The fungus remains intercellular in the cortex but is intracellular in the woody tissue. Ray cell walls are penetrated mechanically, but the xylem through pits. The time taken for various stages of infection to occur is assessed. The amount of damage done by the fungus to roots and the blocking of xylem vessels by tyloses suggest that yellowing, curling and buckling of leaves on infected trees are drought symptoms and not a reaction to fungal toxins. The host reacts to the invasion of the cortex by forming a cork cambium and to the invasion of the woody tissue by blocking individual cells with phenols and resins, which could be important when breeding disease resistant Hevea root stocks.     

Powdery mildew of tobacco (Erysiphe cichoracearum DC.): Susceptibility of proximal and distal parts of leaves from different stalk positions on intact and topped field plants in relation to free amino nitrogen and carbohydrate content*

The area covered by visible mycelium of E. cichoracearum on the upper surface of leaves 4, 8, 12 and 16 of tobacco plants in field plots in Rhodesia was expressed as percentages of the proximal and distal halves at weekly intervals. Free amino nitrogen and carbohydrate in discs from proximal and distal halves of the same leaves were analysed when each leaf was expanding rapidly and was not infected, and several weeks later, when the rate of expansion had slowed down and there was slight infection. On two other occasions, similar leaf discs were inoculated with conidia, to measure the percentage germination and hyphal length from individual conidia after incubation for 2–3 days at constant temperature and humidity; duplicate discs were chemically analysed. Leaves were not susceptible until at least 6 weeks after they had emerged from the bud. Soluble carbohydrate increased and free amino nitrogen decreased during the change from resistance to susceptibility. Proximal parts of leaves were usually infected first; they initially contained less amino nitrogen and soluble carbohydrate than distal parts. All parts of the leaf seemed to be equally susceptible later, when there were no differences in their amino nitrogen or soluble carbohydrate. Upper leaves of intact plants had more natural infection than those from corresponding leaves from topped plants. More conidia germinated on discs from them and produced longer hyphae. The discs from intact plants contained less free amino nitrogen and more soluble carbohydrate than those from topped plants. The accuracy of visual assessments of susceptibility was, generally, confirmed by measurements of percentage germination and length of hyphae from individual conidia on leaf discs. Regressions of hyphal length on leaf composition showed that susceptibility was apparently related inversely to free amino nitrogen and water content and directly to insoluble carbohydrate per unit dry matter.     

The biology of Peronospora viciae on pea: factors affecting the susceptibility of plants to local infection and systemic colonization

Peronospora viciae (Berk.) Casp. penetrated leaf disks of Pisum sativum L. through the cuticle. Resistance of pea plants and of individual leaves to infection by P. viciae increased with age, but decreased again at senescence. Resistance was shown by a restriction in fungal growth and sporulation and by a chlorotic reaction in the leaves. Systemic invasion followed infection of meristematic tissue, and was induced by inoculation into the apical bud of young plants, or on to the epicotyl or hypocotyl, but not roots of germinating seedlings. Most plants whose growth was retarded showed an increased resistance to systemic infection. Pods were infected externally by sporangia, rather than by mycelial growth through the peduncle and pedicel. Oospores and mycelium were found in the testas of some seeds, but seeds from infected pods did not give rise to infected seedlings.     

Germination of Erysiphe gratninis f.sp. hordei conidia on barley leaves

Germination of Erysiphe graminis f.sp. hordei conidia on leaves of several barley cultivars was studied in the laboratory. On both detached leaves and intact plants, within 48 h of inoculation a higher proportion of conidia had germinated on the basal and middle portions of the adaxial leaf surface than on the corresponding portions of the abaxial surface. Such differences between surfaces were not observed near the leaf tip. Similar results were obtained with all the cultivars and growth stages tested, and with five isolates of E. graminis, and are consistent with the observation that there is usually less powdery mildew on the abaxial than the adaxial surface of barley leaves. With most of the barley genotype/mildew isolate combinations tested, within 48 h of inoculation higher proportions of conidia germinated on seedlings and juvenile plants than on older plants. Inherited characteristics which affect spore germination on the leaf surface may be important factors in the development of adult-plant resistance of barley to powdery mildew, particularly in certain genotypes.     

Molecular mapping of genes conferring field resistance to South American Leaf Blight (<Emphasis Type="Italic">Microcyclus ulei</Emphasis>) in rubber tree

The South American Leaf Blight (SALB), caused by the fungus Microcyclus ulei, is the major rubber tree disease in all Central and South America. A population of 192 progeny individuals derived from a cross between a resistant clone and a susceptible cultivated clone was planted in a field trial in French Guiana in order to evaluate the resistance parameters under real infestation conditions. The resistance type (RT), the presence of stromata (ST) and the level of attack (AT) were observed 20-times on a 22-months period, and semi-quantitative evaluation of stromata was registered only once. The search for QTLs was performed using the Kruskal-Wallis test, Interval Mapping and the Composite Interval Mapping method. One major QTL located on linkage group g13 was detected on the RO 38 map, responsible for 36 to 89% of the phenotypic variance of resistance. This resistance QTL corresponds to one that had previously been detected under controlled conditions of infestation and we called it M13-1bn. Surprisingly, the effect of this QTL was larger under natural conditions of infestation than under controlled inoculation. Other minor QTLs (four on the RO38 map and one on the PB 260 map) were also detected. The type of resistance brought by M13-1bn, as well as its durability, are discussed. Applications for rubber tree breeding programs are considered.Communicated by C. Möllers