Asexual Recombinants of Plasmopara halstedii Pathotypes from Dual Infection of Sunflower

Abstract

Genetically homogenous strains of Plasmopara halstedii differing in host specificity and fungicide tolerance were used to test the hypothesis that asexual genetic recombination occurs and may account for the high genotype diversity of this homothallic reproducing oomycete, which causes downy mildew in sunflower. Dual inoculation of sunflower seedlings with single zoospore strains of complementary infection characteristics caused sporulation under conditions where inoculation with each strain alone failed to infect. PCR-based investigation with strain-specific primers proved the presence of genetic traits from both progenitors in single sporangia collected from sporangiophores of such infections. Sister zoospores released from these sporangia revealed the genotype of the one or the other parental strain thus indicating heterokaryology of sporangia. Moreover, some zoospores showed amplification products of both parents, which suggests that the generally mononucleic spores derived from genetic recombination. The possibility of parasexual genetic exchange in the host-independent stage of infection and the evolutionary consequences are discussed.

Fig 1. Theoretical modes of parasexual genetic exchange in Plasmopara halstedii within host-independent (A-C) and host-dependent (D) stages of infection.

A, uptake of nucleus from bursted zoospore of strain I into zoospore of strain II (as proposed by Gu and Ko [16]); B, zoospore fusion (as suggested by Ersek et al. [15]); C, fusion of germtubes from sporocysts; D, hyphal anastomosis (as proposed by Hammer and Spring [17]). Heterokaryon situation (black and white nuclei) may finally lead to recombination (grey nuclei) and result in sporangia with uniform parental or mixed genotype as well as monokaryotic zoospores of either strain I or strain II or a new recombinant type.

Fig 2. Amplification products of single sporangia.

Single sporangia (lane 1–16) were collected from infected plants of the dual inoculation experiments with strain A+B (upper part) and A+C (lower part) and PCR products analysed by capillary electrophoresis. A/B: Lanes 1, 13, 14 show the 113 bp product characteristic for strain A; lanes 2, 7, 8 show the 165 bp product of strain B; lanes 4, 6, 14 show products of both progenitors in different amounts; lanes 3, 10, 11, 12, 16 gave no products. *, negative control with water; L, kb ladder; in the second experiment 7 lanes showed the band of either strain A (5, 6, 7, 9) or C (2, 4, 14), while 3 lanes (1, 3, 8) displayed a combination of both progenitors and 5 lanes (10, 11, 12, 15) failed to give products.

Fig 3. Amplification products of single zoospores.

Zoospores (lane 1–8) were collected from a single sporangium of infected plants of the dual inoculation experiments with strain A+B and analysed by capillary electrophoresis. Lane 2 shows the 113 bp product characteristic for strain A; lanes 4 and 7, contain the 165 bp product of strain B; lane 1 displays both products; lanes 3, 5, 6 and 8 gave no amplicons. *, negative control with water; L, kb ladder.