An Improved Method for Infecting Tomato Leaves or Seedlings with Oospores of Phytophthora infestans Used to Investigate F1 Progeny

Abstract

An improved, soil-free laboratory method was developed to induce late blight infection in detached tomato leaves or tomato seedlings with in vivo-produced oospores of Phytophthora infestans. Oospores were produced in detached tomato leaves infected with an inoculum mixture of A1 and A2 isolates. The infected leaves were homogenized in distilled water. The homogenates were exposed to two cycles of drying and wetting to kill sporangia, zoospores, and mycelia and, thereafter, mixed with perlite and water. Tomato leaves or seed were floated on the perlite mixture in petri dishes and incubated for up to 30 days. Results from 13 experiments showed that, in dishes containing oospores, 96 of 820 leaflets (11.7%) developed late blight lesions within 6 to 30 days; whereas, in 3 experiments with seedlings, 12 of 1,400 plants (0.9%) were blighted within 17 to 26 days. No late blight developed in leaflets or seedlings maintained in dishes containing homogenates of the control leaves infected with either A1 or A2 inoculum. In all, 234 single-sporangium F1 isolates recovered from 24 leaf lesions produced by four crosses and 27 isolates recovered from 12 seedlings infected with two crosses were examined for sensitivity to metalaxyl and mating type. Results showed that, although the parent isolates were either S (sensitive) or R (resistant) to metalaxyl, most F1 progeny isolates exhibited various levels of intermediate resistance to metalaxyl. Most isolates belonged to either the A1 or the A2 mating type, depending on the cross and lesion. However, some isolates belonged to the unusual mating type A1A2 or were sterile. Current experiments are intended to elucidate the virulence and aggressiveness of these F1 progenies to tomato. This improved method may facilitate our understanding of the role of in vivo-produced oospores in the epidemiology of late blight in tomato.

Fungicide modes of action and resistance in downy mildews

“…Since for P. viticola, no other data on resistance segregation in the F1 are available in the literature, it remains unclear whether the genetic background of mefenoxam resistance is different in P. viticola compared to P. infestans, or whether sexual recombination and oospore production follows some yet unknown modifications in P. viticola as was speculated to occur for the inheritance of QoI resistance (see above). Similarly, a distorted segregation pattern was also described in P. infestans when oospores were produced in planta as compared to in agar cultures (Van der Lee et al 2004;Rubin and Cohen 2006). In addition, the segregation pattern of phenylamide resistance in P. viticola was described for F2 progeny by Gisi et al (2007b): a proportion of s:i:r=1:2.7:2 was observed which was considered to be based on one semidominant gene affected by minor genes as described for P. infestans (Judelson and Roberts 1999).…”

“…After 14 days of incubation, plenty of oospores were produced in the leaves which were further incubated in dry conditions in the dark for another 8 weeks. Then, the rotted leaves with oospores were ground to powder, mixed with perlite and moistened with water for inducing oospore germination (Gisi et al 2007b) as originally described for P. infestans (Rubin and Cohen 2006). Young grape leaves were incubated on top of the oospore/perlite mixture for 1 to 3 weeks until first sporangiophores appeared which were picked and propagated for producing F1 progeny isolates.…”

Mating type and sexual reproduction of Pseudoperonospora cubensis, the downy mildew agent of cucurbits

“…After fixation, the cells Inoculation with oospores Leaves containing oospores were homogenized in ice-cold distilled water (10 ml per leaf). To differentially kill the vegetative structures of the pathogen (sporangia, zoospores, cystospores and mycelium), the homogenate was poured into 9-cm Petri dishes (5 ml per dish) and exposed to two cycles of drying and wetting (7-15 h per cycle at 23-28°C), a procedure known to be lethal to vegetative structures of P. cubensis and P. infestans (Cohen et al 1974) but not to their oospores (Rubin and Cohen 2006). After the second drying period, the homogenate was re-suspended in distilled water (3 ml per leaf homogenized) and inoculated onto detached leaves of various cucurbits, lying lower surface uppermost, in Nunk plastic boxes, 25-50 droplets (25 μl each) per leaf inoculated.…”

Inheritance of resistance to carboxylic acid amide (CAA) fungicides in Plasmopara viticola

“…Afterwards, 10 -15 dry leaf discs were homogenized in 5 mL cold distilled water; the oospore concentration was about 500 -1800 mL − 1 . The homogenate was diluted with 7·5 mL water and mixed with 2 g ground perlite in 9-cm-diameter Petri dishes as described by Rubin & Cohen (2006) for Phytophthora infestans . For germination of oospores and infection with P. viticola , very young grape leaves were floated on the perlite/oospore mixture and plates incubated for 7-10 days at 19 ° C in a growth chamber (14 h light per day).…”