Detailed results are given as Electronic Supplementary Material (ESM 1). Detached-leaf assay The C. cassiicola isolates were cultivated on PDA at 25 °C with a 12 h photoperiod. The conidia were collected and resuspended in sterile water supplemented with 0.02 % Tween20 at a concentration of 5000 conidia/ml. For each Go6983 mw isolate, six leaves were inoculated,

each with ten drops of 20 μl conidia suspension applied to the abaxial surface of detached rubber tree leaflets in developmental stage C (brownish to limp green) (Hallé and Martin 1968). One additional drop of 20 μl of sterile water supplemented with 0.02 % Tween20 was added to each leaflet as negative control. The leaflets were maintained in a moist environment at 25 °C for 24 h in the dark and then under alternate light with a 12 h photoperiod. The conidial suspension was evaporated four days after the inoculation.

The lesion area per leaflet was measured manually, at 5 and 9 dpi. The entire experiment was conducted three times. The symptoms intensity (SI) was expressed as the mean lesion area ± the standard error from the 18 inoculated leaves (six leaflets per inoculation and three biological selleck chemical replicates). Detection of cassiicolin gene Wortmannin homologues Detection of cassiicolin gene homologues by PCR was conducted on the four C. cassiicola isolates (E70, E78, E79 and E139) from asymptomatic mature rubber tree leaves. The first set of primers was designed from the Cas sequence from isolate CCP (EF667973) and included CasF9, CasF11, CasF12, CasR16, CasR20 and CasR19. The second set of primers, CT1F9, CasF14, CT1R16 and CasR22, was designed from the CT1 sequence from the isolate Carbohydrate CC004 (GU373809). Primer sequences are listed in the Electronic Supplementray Material ESM 2. PCR was performed on 100 ng of C. cassiicola genomic DNA for 30 cycles

(45 s at 94 °C, 45 s at 50 °C, 45 s at 72 °C) using the same PCR components described above. Cloning of full-length Cassiicolin gene homologues The full-length sequence of the cassiicolin gene homologue Cas3 was obtained by genome walking (Sallaud et al. 2003). This method allows for amplification of the 5′ and 3′ flanking regions of a target gene. Genomic DNA from isolate E70 was digested with 30 units of a restriction enzyme generating 3′ blunt overhangs. Four restriction enzymes were tested independently: EcoRV, DraI, PvuII and StuI (New England Biolabs). The digested products were purified using the QIAquick PCR Purification Kit (Qiagen, Courtaboeuf, France) and ligated to the ADPR1/ADPR2 adaptor by T4 DNA ligase at 16 °C overnight in a final volume of 20 μl. The first PCR was performed with 1 μl of the ligation/digestion using the primer AP1, which is specific to the ADPR1 adaptor, and a primer specific to the Cas3 partial sequence obtained previously from isolate E70 using the CasF9/CasR20 primer pair.