Laboratory tests to assay responses of rubber (Hevea brasiliensis) genotypes to Phytophthora meadii

Variability in response to Phytophthora infections by different rubber (Hevea brasiliensis) genotypes is attributed to biochemical reactions occur in petioles upon infections. Latex is the main product of the rubber tree which is present in all tissues possibly contributing to the biochemical responses. Latex serum from tolerant genotypes significantly stimulated germination of P. meadii zoospores, while that from susceptible genotypes did not stimulate. Extracts from P. meadii-infected petioles of tolerant genotypes significantly reduced P. meadii zoospore germination, while Myelial growth on liquid medium was significantly inhibited by the healthy petiole extracts of tolerant genotypes. The study explored the possibility of using these criteria for laboratory assay of new rubber genotypes to P. meadii.


Introduction
Hevea brasiliensis (A. Juss.) Muell. Arg. (rubber tree) is infected by several pathogens and the major pathogens include Phytophthora meadii McRae and other species, Corynespora cassiicola (Berk & Curt.), Colletotrichum gloeosporioides (Penz.) Sacc., C. acutatum Simmonds ex Simmonds, Oidium heveae Steinm. Immature rubber plants in nurseries are susceptible to most of the above pathogens, whereas mature trees in plantations resist some of the pathogens such as C. cassiicola. The tolerance of one genotype to a certain disease is a unique genetic trait. Therefore, defencerelated biochemical factors are important to be evaluated towards building up a relation to the resistant level of genotypes to P. meadii.
Although mechanisms of plant resistance are still not fully understood, it is known to be under genetic control (Kombrink & Somssich, 1997). Amongst many resistance related activities, synthesis of PR proteins is an important plant defence mechanism (Kombrink & Somssich, 1995), while Phenylalanine Ammonia-Lyase (PAL) and oxidase activities are indicators of host resistance (Narasimhan et al., 2000). Cinnamyl-alcohol dehydrogenase and isoperoxidases are also known as important in plant resistance since, they were found as increased amounts in infected rubber roots (Nicole et al., 1985) while, Scopoletin was observed in leaves infected by C. gloeosporioides (Giesemann et al., 1986). PR-proteins (Narasimhan et al., 2000) and several phenolics in petioles (Jayasuriya et al., 2003) have also been related to resistance of rubber to P. meadii. However, formation of lignin has been noticed as an important tolerant reaction of the resistant genotype RRIC100 upon P. meadii infection. Vanillin was also found as prominent phenolic substance in petioles of RRIC100 (Jayasuriya et al., 2003).
The rubber genotype RRIC 121 which is susceptible to Phytophthora leaf fall disease but resist bark infection caused by the same pathogen (Jayasinghe & Wettasinghe, 1997). This suggests the involvement of many factors in the resistance response of rubber against Phytophthora. This paper explores the possibility of using some biochemical responses involved in the resistance of rubber to selected P. meadii strains, for the development of a laboratory assay to determine the level of resistance of rubber genotypes to P. meadii.

Inoculation of petioles with P. meadii
Mature petioles were obtained from five-year-old trees of each genotype and the cut ends were sealed off with molten paraffin. Thereafter, the petioles were surface sterilised, kept in plastic trays and inoculated with a zoospore suspension (10 4 zoospores ml -1 ) obtained from the isolates MAD86 or DF600 and incubated at 272C for 72 h as described previously (Jayasuriya et al., 1999).

Determination of the effect of latex serum from different rubber genotypes on germination of P. meadii zoospores
The effect of latex serum against P. meadii was tested to determine if any relationship exist between latex and petiole infections. Latex was obtained during early hours of the day. Latex collected (10 ml) from at least 5 field trees (4-year-old) was centrifuged at 15,000 g for 30 min (Beerhues et al., 1994) in 1.5 ml microfuge tubes. Serum was obtained using a syringe and filtered through a Millipore filter (Nalgene  , 0.2 m) and used immediately or stored at -20ºC until use. This serum is referred in the text as sterilised serum.
A 2.9 ml of zoospore suspension (10 4 zoospores ml -1 ) from MAD86 was mixed with 0.1 ml of sterilised serum in McCartney bottles at 272C. Zoospore germination was suspended after 1.5 h by adding 1 drop of Cotton Blue in lactophenol to each bottle. A zoospore suspension similarly mixed with sterilised distilled water served as the control. Drops of the suspensions were thereafter mounted on glass slides and the germination of randomly selected 25 zoospores was microscopically (×100) assessed. Zoospores having germ tubes longer than their breadths were considered as germinated. Four replicate slides were prepared for each suspension and the experiment was repeated twice and results were pooled.

Determination of the effect of extracts from healthy or infected rubber petioles on P. meadii
This investigation was carried out to extract soluble antifungal phenolic compounds from healthy and infected rubber petioles. Excised petioles (obtained from the top whorls of at least 10 trees of each genotype) were immediately washed with sterile distilled water and the excess water was drained off. Small pieces cut from the middle portions (40g) were homogenised with 50% (v/v) boiling ethanol (Harborne, 1989). The homogenate was kept overnight at 4C and thereafter centrifuged at 3000 g for 10 min. The supernatant was filtered and the residue was re-extracted. The pooled filtrate was dried by rotary evaporation and the residue re-dissolved in absolute ethanol was sterilized by Extracts were used immediately or stored at -20 C until use.
Zoospores from MAD86 were obtained from cultures as described previously (Jayasuriya et al., 1999).
Test extract (1 l) was added to 30 l of sterilised distilled water containing 10 2 zoospores ml -1 on a sterilised glass slide. The slide was incubated for 1 h in a closed Petri plate at 26 C and a drop of cotton blue in lactophenol was added.
Fifty randomly selected zoospores were observed for germination under  100 magnifications. In the control, 1 l of absolute methanol was used instead of the test extract. Assessments were repeated 5 times using 500 zoospores in each instance.

Determination of the effect of petiole extracts on the growth of P. meadii on PDA
Growth was examined in pea broth (De Cock et al., 1992) using the method described by Yoshikawa (1978) after modification. One ml of the test extract was added to 30 ml of pea broth in a 125-ml conical flask. The broth was inoculated with one mycelial disc (5 mm) obtained from the edges of an actively growing P. meadii cultures. Thereafter, the flasks were incubated at 272C for 8 days after which the mycelia were harvested by vacuum filtration. Mycelia were oven-dried at 80C and weighed and the inhibition of the growth was defined as the loss of dry matter against the controls. The control was grown in 31 ml pea broth. Results were expressed as mg of mycelium per 31 ml of medium. Experiments were repeated at least 3 times employing at least 8 replicates each time.

Effect of latex serum on the germination of P. meadii zoospores
Latex serum from resistant genotypes significantly (P<0.05) stimulated zoospore germination (by 142-177 %), while serum from susceptible genotypes either did not increase (in RRIC121 and PB86) or significantly (P<0.05) reduced (in RRIM600) zoospore germination compared to the control (Table 1).

Effect of the extract from healthy petioles on germination of P. meadii zoospores
Test extracts from healthy, resistant genotypes significantly (P<0.05) inhibited germination of P. meadii zoospores than extracts from the susceptible genotypes. The OD 380 values of the extracts from healthy petioles of RRIC100 and BPM24 were equal and also higher than the OD 380 values of similar extract of susceptible genotypes ( Table 2).

Effect of extracts from P. meadiiinfected petioles on germination of P. meadii zoospores
Extracts from infected petioles of resistant genotypes have significantly (P<0.05) inhibited germination of zoospores ( Table 2).

Effect of petiole extracts on growth of P. meadii
The effect of petiole extracts on the growth of P. meadii was not always consistent. Extracts from healthy petioles of susceptible genotypes significantly (p<0.05) increased the mycelial growth in the liquid medium. However, the extracts from infected petioles of the same group did not increase the growth. In majority of cases, the effect on the growth of was significantly (p<0.05) lower when the medium was amended with the extract from infected petioles (Table 3).

Discussion
Due to the non-availability of more clones established either as resistant or susceptible to diseases caused by Phytophthora, only five rubber genotypes were used in this investigation. The susceptible genotypes used in the study had similar characteristics, which significantly varied from the characters of the tolerant genotypes. Latex serum from tolerant genotypes had pronounced effect on zoospore germination which probably can be attributed to sugars or proteins in the serum as sugars and proteins are known to promote zoospore germination of Pythium spp (Donaldson & Deacon, 1993). However, although a variation among the total protein content in serum of rubber clones was not observed, a difference in basic protein pattern had been reported . Therefore, the serum test would be reliable to assay the difference between tolerance and susceptible genotypic effect against P. meadii.
The toxic reaction of tolerant genotypes against P. meadii is probably due to different phenolic substances contained in petioles. The tolerant types are reported to contain more fungitoxic substances such as vanillin (3-methoxy-4-hydroxybenzaldehyde) (Jayasuriya et al., 2003) and other coumarins (Gieseman et al., 1986). The lower effect of extracts from P. meadii infected petioles may be due to polymerization of toxic phenolic monomers upon infection and the formation of insoluble compounds such as lignin. This is apparent from lower OD 380