A. Drenth

Historical and recent migrations of Phytophthora infestans: chronology, pathways, and implications.

The 1984 report of A2 mating types of Phytophthora infestans (Mont.) de Bary in western Europe (20) was the first indication of new and dramatic developments in populations ofthat Fungus. This discovery stimuiated plant pathologists aH over the world to analyze local populations, since previously only the A 1 mating type had been detected outside of central Mexico (Fig. I). The analyses of a large number of dispersed Eocal populations indicated, surprisingly, that the changes were not restricted to western Europe but, rather, were worldwide (Fig. 2) [3,10,23,26,36,41). The recent wortdwide changes in populations mOSK certainly result from migration. Indeed, migration has played an essential role in the entire history of potato late blight. In this article we illustrate that role. To provide context, however, we first present background concerning the basic biology/pathology of P. infestans, the genetic tools used to investigate populations of P. infesrons, and the char. acteristics of the source population of P. infestans.

DNA fingerprinting uncovers a new sexually reproducing population of Phytophthora infestans in the Netherlands.

The oomycetous fungusPhytophthora infestans (Mont.) de Bary, which causes late blight disease in potatoes, is heterothallic with two known mating types, A1 and A2. From 1845 until 1980 only A1 mating type isolates were found in Europe. In 1980, the A2 mating type appeared permitting sexual reproduction. Here we show that virulence properties and DNA fingerprint patterns of isolates collected in the Netherlands before and after the appearance of A2 mating type isolates are different. Before 1980, eight different races were found in which virulence factors 1, 2, 3, 4 and 10 were most common. After 1980, new virulence factors (i.e. 5, 6, 7, 8 and 11) showed up and the diversity for virulence increased tremendously: 73 different races were detected among 253 isolates analyzed. DNA fingerprint analyses of isolates collected before 1980 revealed that, for at least two decades, only one RG-57 fingerprint genotype was present in Europe. Among 179 isolates collected after 1980 134 distinct RG-57 fingerprint genotypes were identified. The dramatic increase in genetic diversity strongly suggests that theP. infestans population in the Netherlands is now propagating sexually. The change from asexual to sexual reproduction, and the resulting increased adaptability and ability to survive outside the host, may interfere drastically with the regular disease control methods.

Evolutionary relationships among Phytophthora species deduced from rDNA sequence analysis

Sequence analysis of the internal transcribed spacer (ITS) regions I and II of ribosomal DNA were used to deduce evolutionary relationships among 15 Phytophthora species. Analysis of papillate, semi-papillate and non-papillate species showed that sporangium papillation has phylogenetic significance, with the three morphological groups each forming separate clusters. Within the P. megasperma species complex, separate evolutionary lines were identified for P. medicaginis, P. trifolii, and P. sojae formerly regarded as formae speciales of P. megasperma, confirming their recent reclassification as biological species. rDNA sequence analysis was able to distinguish P. cryptogea and P. drechsleri isolates indicating a valid basis for speciation. P. macrochlamydospora from soybean, which has only been observed in Australia, was closely related to P. sojae, indicating possible common ancestory.

Phytophthora sojae Avirulence Genes, RAPD, and RFLP Markers Used to Construct a Detailed Genetic Linkage Map

Two crosses between different races of Phytophthora sojae were established using one race as a common parent in both crosses. F2 populations comprising over 200 individuals were generated for each cross. A subset of 53 F2 individuals from each cross was selected at random for genetic analysis of virulence/avirulence and molecular markers, and finally the construction of a detailed genetic linkage map. The linkage map developed for P. sojae is based on a total of 257 markers (22 RFLP, 228 RAPD, and 7 avirulence genes). The linkage map comprises 10 major and 12 minor linkage groups covering a total of 830.5 cM. Close linkage was observed between Avr4 and Avr6 (0.0 cM), Avr1b and Avr1k (0.0 cM), and Avr3a and Avr5 (4.6 cM). Coupling phase linkage of RFLP and RAPD markers to all seven avirulence genes was identified at the minimum and maximum distances of 0.0 and 14.7 cM, respectively.

Origin of the A2 mating type of Phytophthora infestans outside Mexico

The first report of the A2 mating (compatibility) type of the potato late blight pathogen, Phytophthora infestans (Mont.) de Bary, outside Mexico was in Europe during 1984 (32) and, since then, the A2 has been found in many parts of the world (19,23). The four most likely hypotheses to explain the occurrence of the A2 mating type outside Mexico are that it (i) was always present, but undetected (58); (ii) was introduced by migration (62); (iii) arose by mutation or mitotic recombination; or (iv) arose by mating type change, either from exposure to fungicides or by induced selfing (39). Among these, the migration hypothesis is the only one with strong scientific support. However, this subject remains somewhat controversial, and alternative explanations for the origin of the A2 mating type of P. infestans outside Mexico still appear occasionally in the literature. Analyses of allozyme data provided the first unambiguous evidence that the A2 mating type in Europe and Japan was introduced by migration from Mexico (62). Numerous additional population genetic studies have fully supported the migration hypothesis (13, 15,18,23,25,40,44,55,63). In each location studied, the first detection of A2 isolates coincided with the appearance of new alleles at allozyme, DNA fingerprint, and mitochondrial DNA loci. Similar changes occurred with the recent appearance of the A2 mating type in the United States (29). Although the detection of new alleles sometimes preceded the A2 (1,25), the A2 never appeared without new alleles (19,23). The migration hypothesis was challenged recently by Ko (39), who proposed instead that mating type change was the origin of the A2 mating type of P. infestans outside Mexico. Ko’s (39) conclusion came from his result that self-fertilization could initiate mating type change. Unfortunately, the mating type change hypothesis in P. infestans was not tested using genetic markers, and no genetic mechanism was proposed by which mating type change could occur. Furthermore, the population genetic data that contradict the mating type change hypothesis were ignored. Part of the proof for the mating type change hypothesis was based on a number of early literature reports that supposedly stated that homothallic isolates of P. infestans were present outside Mexico prior to the 1950s. Unfortunately, these early references were cited without critical evaluation. It is well-known that heterothallic species of Phytophthora produce occasional oospores in single culture (4,21,53,64, 65). It is also quite well documented that oospores of P. infestans were found occasionally during the early part of this century in Europe and the United States (8,48). However, because these structures were produced only rarely and under specific conditions, the scientific consensus was that the sexual stage of P. infestans remained to be discovered. This did not change until the A2 mating type was found in Mexico during the 1950s (21,47,61). Because of the strong evidence for migration, the mating type change hypothesis has never been tested directly. Fortunately, this hypothesis provides testable predictions about the genetic background that should be present in A2 isolates outside Mexico. If the A2 originated by mating type change from A1 mating type populations, the first A2 isolates in each location should be identical, or nearly identical except for mating type, to the previously existing A1 isolates. Sexual reproduction after mating type change could generate new genotypes, but they still should contain only the alleles present in the original A1 populations. Because most populations throughout the world, until recently, were composed primarily, or exclusively, of a single clonal lineage (15,23,25), the mating type change hypothesis is easily testable using molecular markers. Identical multilocus genotypes (or changes limited to a rearrangement of alleles) before and after the occurrence of the A2 mating type would confirm the mating type change hypothesis. In contrast, if the A2 mating type originated by migration, A2 isolates could be similar, or very distinct, from the original A1 isolates, depending on the source population for the migrating genotypes. If the first A2 isolates were very different from isolates in the old A1 populations, the mating type change hypothesis would be rejected. Our purpose was to reanalyze previously published genotypic data to explicitly test the mating type change hypothesis for the origin of the A2 of P. infestans outside Mexico. A secondary goal was to evaluate the early literature to test Ko’s (39) conclusion that homothallic isolates of P. infestans were known outside Mexico prior to the 1950s. Finally, mating type segregations in self-fertilized progenies of P. infestans were analyzed to determine whether mating type change has been observed by other investigators.

Evidence for outcrossing in Phytophthora sojae and linkage of a DNA marker to two avirulence genes

Two genetically different isolates of the homothallic Oomycete, Phytophthora sojae, were demonstrated to outcross and form hybrid oospores after co-culturing in vitro. Random amplified polymorphic DNA (RAPD) markers revealed ten hybrids among 354 oospores analysed. One F1 hybrid was allowed to self fertilise and produce an F2 population of 247 individuals. Among 53 F2 individuals, selected at random, 18 polymorphic RAPD markers were observed to segregate at near 3:1 Mendelian ratios, consistent with segregation for dominant alleles at single loci. Segregation of virulence against soybean resistance genes Rps1a, 3a, and 5 revealed that the avirulence genes Avr1a, 3a and 5 were dominant to virulence. Avirulence against these three resistance genes appeared to be conditioned by one locus for Avr1a and two independent, complementary dominant loci for both Avr3a and Avr5. Segregation of virulence against Rps6 was in the ratio of 1:2:1 (avirulent:mixed reaction:virulent), suggesting a semi-dominant allele at a single locus. Two avirulence genes and one RAPD marker formed one linkage group, in the order Avr3a, OPH4-1, Avr5, each separated by approximately 5 cM. Our results confirm that outcrossing occurred between the parental isolates, and that sexual recombination under field conditions may play an important role in generating and maintaining genetic diversity in populations of P. sojae.

The occurrence of the A2 mating type of Phytophthora infestans in the Netherlands; significance and consequences

Phytophthora infestans (Mont.) de Bary, the causal agent of potato late blight, was first discovered in Europe in 1845. Until 1980, only A1 mating type isolates were known to occur in Europe. The absence of A2 mating type isolates restrained the fungus from sexual reproduction. In the early 1980s, A2 mating type isolates were discovered in Europe. Presumably, a new introduction ofP. infestans isolates originating from Mexico had taken place. In this paper, the significance of the presence of A1 and A2 mating type isolates in the Netherlands is reviewed. Now that both mating types are present, sexual reproduction can occur and its consequences for the control of potato late blight are discussed.

Inheritance and mapping of 11 avirulence genes in Phytophthora sojae.

Two new crosses involving four races (races 7, 16, 17, and 25) of the soybean root and stem rot pathogen Phytophthora sojae were established (7/16 cross; 17/25 cross). An F2 population derived from each cross was used to determine the genetic basis of avirulence towards 11 different resistance genes in soybean. Avirulence was found to be dominant and determined by a single locus for Avr1b, 1d, 1k, 3b, 4, and 6, as expected for a simple gene-for-gene model. We also observed several cases of segregation, inconsistent with a single dominant gene being solely responsible for avirulence, which suggests that the genetic background of the different crosses can affect avirulence. Avr4 and 6 cosegregated in both the 7/16 and 17/25 crosses and, in the 7/16 cross, Avr1b and 1k were closely linked. Information from segregating RAPD, RFLP, and AFLP markers screened on F2 progeny from the two new crosses and two crosses described previously (a total of 212 F2 individuals, 53 from each cross) were used to construct an integrated genetic linkage map of P. sojae. This revised genetic linkage map consists of 386 markers comprising 35 RFLP, 236 RAPD, and 105 AFLP markers, as well as 10 avirulence genes. The map is composed of 21 major linkage groups and seven minor linkage groups covering a total map distance of 1640.4cM.

Pythium insidiosum keratitis confirmed by DNA sequence analysis.

Editor,— Pythium insidiosum is an unusual but serious ocular pathogen. Although the organism grows as a mycelium in tissue, it is not a member of the fungal kingdom and its identification can be a challenge for a routine laboratory. We report a case of Pythium keratitis in which the organism was confirmed by nucleic acid sequencing. ### CASE REPORT A 32 year old man was referred from Kuala Lumpur having suffered with intractable keratitis of the left eye for 4 weeks. He gave a history of diabetes, disposable contact lens wear, and swimming in the Kelang River. Routine microbiological investigations had been negative. At presentation to Flinders Medical Centre, he was on topical antibacterial, antifungal, and antiamoebic medication. He had a large epithelial defect, a deep stromal infiltrate approaching the limbus, and hypopyon (Fig 1, top). His visual acuity was hand movements and there was considerable pain. The drops were stopped and corneal scrapings were taken. Gram and Giemsa stains were negative. A biopsy was performed the following day and hyphae were observed in sections. A filamentous organism appeared in cultures of the original scrapings. There was no response to continued antifungal treatment and a penetrating graft was performed 4 days after the biopsy. Postoperatively, the patient received oral itraconazole and …

Phytophthora in Australia

Phytophthora spp. are one of Australias most serious plant pathogens, causing well in excess of