A2 Compatibility Type Research Articles

First Report of Nothofagus macrocarpa Dieback Caused by Phytophthora citrophthora and P. nicotianae in Chile.

The genus Nothofagus, family Nothofagaceae, comprises 36 species of trees that are native to the Southern Hemisphere. N. macrocarpa (DC.) F.M. Vásquez & R.A. Rodríguez (Roble de Santiago) is an important deciduous tree, endemic to central Chile (32 to 35°S), and found above 800 m altitude. There is an increasing interest in N. macrocarpa as an ornamental. However, a general dieback (40 to 50% prevalence) was observed at a commercial nursery in Santiago in 2009, limiting its multiplication. Symptoms are wilting, partial defoliation, reddish brown cankers on the crowns, and root necrosis. The purpose of this work was to study the etiology of the dieback in nurseries. Phytophthora was isolated from the roots and cankers of symptomatic plants (n = 3) and soil samples (using apples and avocados as baits) on amended corn meal agar (3) at 20°C for 5 days in the dark. Morphologically, P. citrophthora (Smith & Smith) Leonian, and P. nicotianae Breda de Haan were identified (2). On V8 juice agar (V8) (1), P. citrophthora formed petaloid colonies, grew between 5 and 30°C (optimum of 25°C), and produced deciduous, mono- or bipapillated sporangia of (28.1) 45.0 to 64.1 × (18.8) 32.0 to 39.2 μm. On V8, P. nicotianae produced cottony colonies, grew between 10 and 30°C (optimum of 25°C), and produced spherical, intercalary chlamydospores (mean diameter of 19.6 μm) and persistent, papillate, spherical to ovoid, ellipsoid, obpyriform sporangia of (33.2) 47.5 to 67.6 × (24.1) 30.0 to 48.9 μm. Isolates of P. citrophthora were sexually sterile, but P. nicotianae formed oogonia with amphigenous antheridia in dual cultures with P. cinnamomi (A2 compatibility type). BLAST analysis of the internal transcribed spacer (ITS) region of rDNA of isolates identified as P. citrophthora (IMI 399056 and IMI 399054, GenBank Accession Nos. JF699756 and JF699755) and P. nicotianae (IMI 399055, Accession No. JF699757), amplified by PCR using ITS universal primers (4), revealed 100% similarity with reference isolates of P. citrophthora (Accession Nos. GU259324.1 and GU259317.1) and P. nicotianae (Accession No. GU983635.1). P. citrophthora (n = 2) and P. nicotianae (n = 1) were pathogenic when wounded detached twigs (n = 5) of N. macrocarpa and N. obliqua were inoculated with 20 μl of a mycelial suspension (106 CFU/ml) of either Phytophthora spp. Twigs were placed in a moist chamber at 20°C for 12 days prior to determine the length of the necrotic lesions that developed. An equal number of noninoculated twigs were left as control. Reisolation of P. citrophthora and P. nicotianae from inoculated material was 100%. The length of the necrotic lesions (13 to 80 mm) from inoculated N. macrocarpa and N. obliqua was significantly greater (P < 0.05) compared with the controls. Regardless of Phytophthora isolates, necrotic lesions (53.9 ± 15.8 mm) in infected N. macrocarpa were significantly longer than in N. obliqua (28.6 ± 13.1 mm) (P < 0.0001). To our knowledge, this is the first report of P. citrophthora and P. nicotianae associated with dieback on N. macrocarpa in Chile. Therefore, there is a potential risk of Phytophthora dieback in N. macrocarpa in nature.

Identification, Mefenoxam Sensitivity, and Compatibility Type of Phytophthora spp. Attacking Floriculture Crops in North Carolina.

Phytophthora isolates were collected from floriculture crops grown in commercial greenhouses in North Carolina for species identification, compatibility type determination, and mefenoxam sensitivity tests. Isolation from 41 symptomatic plant species at 29 production locations resulted in 483 isolates from eight crops at seven locations. Phytophthora cryptogea (184 isolates) was recovered from dusty miller and gerbera daisy. All isolates of P. cryptogea were insensitive or intermediate in sensitivity to mefenoxam at 1 μg a.i./ml and were A1 compatibility type. P. nicotianae (273 isolates) was isolated from African violet, lavender, pansy, petunia, and vinca. Of these isolates, 21% were insensitive to mefenoxam at either 1 or 100 μg a.i./ml. Isolates of P. nicotianae from five locations were A2 compatibility type, whereas isolates on pansy at one location were A1 compatibility type. English ivy grown at two locations was infected with P. palmivora. All 26 isolates of P. palmivora were sensitive to mefenoxam and were A1 compatibility type. Mating type and mefenoxam sensitivity were uniform among isolates of P. nicotianae and P. palmivora from a given crop at a given location, suggesting that epidemics within a location may have originated from a single source of inoculum.

PHYTOPHTHORA NICOTIANAE PATHOGENIC TO PEPPER IN NORTHWEST SPAIN

SUMMARY Seven strains of Phytophthora nicotianae were isolated from pepper plants (Capsicum annuum) showing collar and root rot in northwest Spain and were identified using standard taxonomic criteria. The pathogenic behaviour of the isolates was studied in Capsicum annuum under greenhouse and growth chamber conditions in comparison with that of Phytophthora capsici. Differences between isolates of the two species and great variation within the strains of P. nicotianae were observed. The genetic compatibility of these strains was studied and the importance of some taxonomic criteria was considered. P. capsici isolates were all A1 while those of P. nicotianae had the A2 compatibility type. Criteria for distinguishing the two species are discussed.

Phytophthora inundata sp. nov., a part heterothallic pathogen of trees and shrubs in wet or flooded soils

A Phytophthora pathogen of trees and shrubs previously designated Phytophthora sp. O-group is formally named as P. inundata sp. nov. P. inundata falls within the P. gonapodyides-P. megasperma major ITS Clade 6, its present nearest known relative being P. humicola. It has non-papillate sporangia, fairly large oogonia (average ca 40 microns) with thick walled oospores, amphigynous antheridia, a distinctive colony type, a high optimum temperature for growth of 28-30 degrees C, fast growth at the optimum, and a high upper temperature limit for growth of ca 35-37 degrees. A study of the breeding system of eight P. inundata isolates showed them to be classically heterothallic with A1 and A2 compatibility types. However some P. inundata A1 x A2 combinations failed to mate even though the same isolates mated successfully with P. drechsleri testers. Others were 'silent' A1s or A2s, unable to produce their own gametangia but able to induce gametangial formation in the opposite sexual compatibility type of another species. This indicates a partial breakdown of the sexual mechanism in the species. Two isolates (one A1 and one A2) were unpredictably and chimaerically self-fertile, suggesting A1 + A2 chromosomal heteroploidy. The association of P. inundata with ponds and rivers and with root and collar roots of trees and shrubs after flooding is discussed.

The Spatiotemporal Genetic Structure of Phytophthora capsici in Michigan and Implications for Disease Management

Root, crown, and fruit rot caused by Phytophthora capsici Leonian is a limiting factor for the production of peppers, tomatoes, and cucurbit crops in Michigan and the United States. Like many species in the genus Phytophthora, P. capsici has the potential for rapid polycyclic disease development from a limited amount of initial inoculum (6). P. capsici produces caducous sporangia that can be spread by wind-blown rain or release 20 to 40 motile zoospores in the presence of free water. The polycyclic phase of disease development is thought to be driven primarily by asexual spore dispersal at a local scale (within and down rows). Sexual reproduction requires both the A1 and A2 compatibility types (CTs) and results in the production of thick-walled oospores. Oospores are thought to serve as the primary survival structure outside of host tissue. Recommended disease management strategies stress the importance of avoiding excess water in the plant rhizosphere by using well-drained fields, conservative irrigation, and planting on raised beds. Additional recommendations include rotation to nonsusceptible hosts for at least 2 years and the use of fungicides. The phenylamide fungicide (PAF) mefenoxam is a systemic compound with high activity against P. capsici and has been used by growers throughout the United States to control P. capsici. Insensitivity to PAF has been reported for a number of other oomycetous organisms (Bremia lactucae, P. infestans, and P. sojae, etc.) and appears to be conferred by a single incompletely dominant gene of major effect (1). Growers in Michigan practicing 2+-year rotation in well-drained fields using an array of fungicidal management tools have experienced significant losses to P. capsici. Michigan is the number one producer of cucumbers for pickling in the United States and it was at the request of grower groups associated with this industry that research into the epidemiology and reproductive biology of P. capsici on cucurbit hosts was initiated. Although many researchers cite oospores as the most likely propagule for survival outside of host tissue, there have been very few investigations specifically aimed at determining the impact of sexual reproduction in natural populations of P. capsici. Our hypothesis was that the sexual stage may play an important role not only in survival but also in the adaptation of P. capsici populations to environmental stresses (e.g., fungicides). Our goal was to perform a comprehensive investigation of the phenotypic and genetic diversity present in P. capsici populations from the major vegetable production regions of Michigan, with the implicit intention of addressing questions concerning epidemiology, reproductive biology, and the durability of currently recommended management strategies.

First Report of Phytophthora nicotianae on Limonium in Europe.

Limonium (statice or sea-lavendar, family Plumbaginaceae) is grown in the Netherlands as a perennial (Limonium sinense) or annual (Limonium sinuatum) crop. Plants have tufted leaves and numerous clustered flowers of different colors and are used for flower arrangements. In August 2000, we received diseased plants of L. sinense cv. Diamond and L. sinuatum. Disease symptoms consisted of leaf wilting followed by plant collapse. The base of the leaves showed progressive necrotic areas that later turned dark brown to black. The cortex of the stem and roots was water-soaked and dark brown to black. Longitudinal sections of stems and roots of diseased plants displayed discoloration of tissues. Rotted root tissue was brown with a characteristic black margin. Rotted vascular tissues and other stem parts were also dark brown. Pith parenchyma turned gray-brown and had a firm, wet rot. In plants with advanced disease symptoms, a cavity in the stem parenchyma was observed. Isolations were made from sections of symptomatic leaves, stems and roots of both Limonium species on cherry and water agar (WA), followed by incubation at 20°C. Phytophthora sp. was isolated consistently from the base of leaves, stems, and roots of diseased plants and identification of isolates was based on morphological characteristics and by isozyme analysis (3). Observations of colony morphology and growth at 35°C were made on V8 agar. Mating type was determined in dual cultures with mating type A2 (P. nicotianae, P 1923 [4]) and A1 (P. nicotianae, PD98/8/10402). Sporangial features were observed from liquid cultures of the isolates (autoclaved soil-extract or sterile distilled water). All isolates formed colonies consisting of loose, fluffy aerial mycelia. Sporangia and chlamydospores were present in all fungal isolates and all isolates were able to grow at 35°C. Few sporangia were produced on solid media (WA and V8 juice agar), but were abundant in liquid cultures. Sporangia were borne singly or in simple sympodial sporangiophores (3 to 4 sporangia), and were ovoid/spherical, obturbinate with rounded base and had prominent papillae (some had two papillae). Sporangia measured 40 to 64 × 24 to 56 μm, (average 50.4 × 38.4 μm) and had an average length:breath ratio of 1.3:1. Chlamydospores were terminal and intercalary and measured 18 to 44 μm (average 31.6 μm). Hyphal swellings with hyphal outgrowths were present. Isolates of the fungus were heterothallic and produced oogonia and oospores rapidly and abundantly on V8 agar at 22°C only with the A1 mating type of P. nicotianae. We concluded that all isolates from Limonium had the A2 compatibility type. Antheridia were amphigynous. Oogonia were spherical and ranged from 20 to 30 μm, (average 27.5 μm). Oospores ranged from 18 to 27 μm, (average 23.1 μm). The observed characteristics are similar to those described for P. nicotianae. Isozyme analysis, using the dimeric enzymes malic enzyme (EC 1.1.1.40) and malate dehydrogenase (EC 1.1.1.37), revealed the presence of the Mdhp100 allele and the Mdh-2100 allele. Both alleles are characteristic for P. nicotianae (3). Based on morphological features and isozyme genotyping, isolates of Phytophthora from diseased Limonium plants could be assigned to P. nicotianae van Breda de haan (1). A report from Florida associated Phytophthora sp. with root rot of Limonium plants (2) but did not identify the species. According to the multi-decade records at the Netherlands Plant Protection Service (unpublished data) Phytophthora has never been observed on Limonium before. This is the first report of P. nicotianae associated with root rot and basal rot of Limonium plants in Europe.

Mefenoxam Insensitivity and the Sexual Stage of Phytophthora capsici in Michigan Cucurbit Fields

ABSTRACT The potential for outcrossing, occurrence of oospores, and inheritance of mefenoxam sensitivity was assessed in naturally occurring populations of Phytophthora capsici. Between 1997 and 1998, 14 farms were sampled, with 473 isolates recovered from cucurbit hosts and 30 from bell pepper. The A1 and A2 compatibility types were recovered in a roughly 1:1 ratio in 8 of 14 farms with sample sizes larger than 15. In 1997, one isolate was designated as insensitive and four as sensitive to mefenoxam. In 1998, 55% of the 498 isolates sampled were sensitive, 32% were intermediate, and 13% were fully insensitive to mefenoxam. In vitro characterization of mefenoxam sensitivity was conducted by crossing field isolates. Chi-square analysis of crosses between sensitive, intermediately sensitive, and insensitive isolates indicate that mefenoxam insensitivity segregated as an incompletely dominant trait unlinked to compatibility type (P = 0.05). Oospores were observed in diseased cucurbit fruit from four farms in 1998, and 223 oospore progeny were recovered from a single diseased cucumber. All six mefenoxam sensitivity-compatibility type combinations were present in these oospore progeny and within single fields. Based on these findings, we conclude that oospores likely play a role in the survival of P. capsici and that sexual recombination may significantly influence population structure.

Characterization of the Phytophthora infestans Population in the Columbia Basin of Oregon and Washington from 1992 to 1995

ABSTRACT Isolates of Phytophthora infestans collected from 1992 to 1995 from potato fields in the Columbia Basin of Oregon and Washington were analyzed for compatibility type, metalaxyl sensitivity, and glucose-6-phosphate isomerase (Gpi) genotype. In 1992, 30 of 31 isolates were of the US-1 multilocus genotype. A single metalaxyl-resistant isolate of the US-6 (A1 Gpi 86/100) genotype was found near the end of the growing season. In 1993, only 2 of the 59 isolates collected were A1 isolates with Gpi 86/100. Ten isolates were of the A2 compatibility type, seven with Gpi 100/111, two with Gpi 100/100, and one was undetermined. The remaining isolates were metalaxyl-resistant A1 compatibility types with either Gpi 100/100 or 100/111. The first A2 isolates in the Columbia Basin were found in 1993. In 1994, 10 of 18 isolates were of the US-1 genotype. The remaining isolates were US-6 and US-8 genotypes. In 1995, 97% of 268 isolates tested were of the US-8 genotype. Five isolates were A2 compatibility type with Gpi 100/122. One A2, metalaxyl-resistant isolate was Gpi 100/100/111, and two A1 isolates were Gpi 100/111/122. The population of P. infestans quickly changed between 1992 and 1995, from a population comprised almost exclusively of the US-1 genotype to a population represented by new or recombinant genotypes.

Phytophthora meadii may be a species hybrid

In a cytological study, some isolates of Phytophthora meadii were found to be predominantly polyploid and to show normal oospore development, while others were predominantly diploid. The diploids produced mainly small oogonia, which degenerated during meiosis with the production of many empty oogonia; but also some larger oogonia with polyploid nuclei and normal oospore development. It is therefore suggested that P. meadii is derived from a species hybrid in which meiosis is upset by the genetical imbalance; and that chromosomal doubling during vegetative growth, followed by selection, leads to the production of tetraploid forms which can undergo normal meiosis. No differences were found in the soluble protein patterns of diploid and polyploid isolates. While both A1 and A2 compatibility types were found in each ploidy group, the polyploid A2's (presumed compatibility type heterozygotes) tended to be weakly self-fertile, and one of them gave rise to a self-sterile polyploid A1 culture. This demonstrates the difficulty of maintaining the heterozygous condition during vegetative growth in a polyploid. The possible role of polyploidy in the apparent decline of the A2 type in Sri Lankan P. meadii populations is discussed.

Relationship Between Inoculum Level ofPhytophthora capsiciand Mortality of Pepper

Relationships between the levels of oospore and zoospore inoculum of Phytophthora capsici and mortality of pepper (Capsicum annuum) were investigated. Oospore progeny from 20 pairings of pathogenic isolates of P. capsici varied in pathogenicity to pepper at an initial inoculum density of 25 oospore per gram of soil. Only oospore inoculum from specific pairings caused disease in growth room experiments. The highest mortality (30-75%) resulted from oospore inoculum produced from pairings involving certain isolates of A2 compatibility type. Oospore inoculum from other pairings caused little or no disease (0-5%) (...)

Phytophthora inpestans A2 compatibility type recorded in Great Britain

The A2 compatibility type of Phytophthora infestans, previously recorded in Mexico and Switzerland, was recognized in two isolates of P. infestans from a crop of potato cv. Wilja grown commercially in Great Britain.

Taxonomy of Phytophthora species isolated from rubber in Sri Lanka

Growth rate on Lima Bean Agar (LBA) colony morphology, morphology and size of sporangiophores, sporangia, sporangial pedicels, chlamydospores, sex organs and frequency of compatibility types were studied for 33 Phytophthora isolates from rubber (Hevea brasiliensis), two from cocoa (Theobroma cacao), P. heveae, P. arecae, P. meadii, P. botryosa, P. megakarya, P. palmivora and Phytophthora MF4 (? = P. capsici). P. meadii was distinguished from P. palmivora by pedicel characters, sporangial and sporangiophore morphology and chlamydospore frequency, size and colour. They differed cytologically and in oospore morphology and size. P. palmivora is distinguished from P. meadii by its ability to produce a brownish tinge in LBA. In P. meadii both A1 and A2 compatibility types occurred with A1 predominant. P. meadii is the principal pathogen of rubber in Sri Lanka and is considered an acceptable species. Of the 33 isolates from rubber 32 were identified as P. meadii and one as P. palmivora. Isolates from cocoa in Sri Lanka also conformed to P. palmivora. A slide culture technique proved particularly useful for identifying different Phytophthora spp.

Mating Types of Phytophthora capsici Leonian, the Causal Fungus of Pumpkin Rot in Hokkaido

Phytophthora rot of pumpkins (Cucurbita maxima, cultivar Ebisu) was observed in Ikeda and Shibetsu, Hokkaido in 1974 and 1975 respectively. Based on morphological, physiological, and pathological characters, the causal fungi were identified as Phytophthora capsici Leonian. All 50 single zoospore cultures of Ikeda isolate behaved as A1 compatibility type. Twenty-eight of 30 single zoospore isolates of Shibetsu fungus, soon after isolation, produced no oospore and behaved as A2 type. But after they were stocked for 18 months (transferred at intervals of 5-6 months), abundant oospores were formed, usuaIly in sector(s) or around initial inoculum when subcultured on V-8 agar plates, incubated at 23C in the dark for 10 days. The other two isolates usually produced oospores in segment(s); one, 15 days after isolation, another, after stocked for 28 months. Sterile areas acted as A2 compatibility type.

STIMULATION OF SEX ORGAN FORMATION IN PHYTOPHTHORA BY ANTAGONISTIC SPECIES OF TRICHODERMA

SUMMARYTwo methods are described for the induction of sex organ formation in heterothallic Phytophthora species in response to Trichoderma, a volatile method and a direct inoculation (‘contact’) method.Tested for their ability to induce sexual reproduction in Phytophthora, isolates of Trichoderma koningii, T. piluliferum, T. polysporum and T. viride were found to be active, and isolates of T. hamatum, T. harzianum, T. longibrachiatum and T. pseudokoningii inactive. Response in Phytophthora was, without exception, confined to the A2 compatibility type. Response of different Phytophthora species, however, was more variable. All forty‐one isolates of P. cam‐bivora and P. cinnamomi tested responded to Trichoderma viride. Isolates of Phytophthora cryptogea, P. drechsleri, and P. nicotianae were apparently polymorphic for response. The black pepper form of P. palmivora responded, whereas P. palmivora forms 1 and 2 did not. A minimum induction period of c. 24 h exposure to volatiles of active Trichoderma was apparently necessary for consistent oogonial formation to occur. Oogonial formation occurred on a range of sterol‐containing media but not in their absence and over a wide range of conditions of temperature and light.Antagonism of Phytophthora cultures by active Trichoderma isolates was readily apparent both in the volatile and contact methods. The ability of Trichoderma isolates to induce a sexual response in Phytophthora was correlated with their ability to produce volatile antibiotics. Possible implications of the ‘Trichoderma effect’ for the taxonomy, reproductive physiology and genetics of Phytophthora spp. are considered.

Oospore production by a single isolate of Phytophthora capsici in the presence of chloroneb

A single isolate of the A2 compatibility type of P. capsici Leonian, a heterothallic species, was induced to form typical oogonia with amphigynous antheridia and oospores in agar cultures by the presence of chloroneb. Production of oospores continued when the cultures were maintained subsequently on medium with or without the chemical. These results suggest that chloroneb has brought about a permanent change in the ability of this isolate to form sex organs. Cultures also produced oospores when exposed to chloroneb vapor.

Comparative Morphology and Behaviour of A1 and A2 Isolates of Phytophthora cinnamomi

Studies of morphology, physiological behaviour and pathogenicity indicated that some significant differences existed between isolates of the A1 and A2 compatibility types of Phytophthora cinnamomi from native plant communities in Australia. The majority of A1 isolates produced zoosporangia more rapidly and in greater numbers than A2 isolates, and zoosporangia were produced evenly on mycelial discs of A1 isolates whereas they occurred mostly at the perimeter of similar discs in the case of A2 isolates. Chlamydospores were produced more rapidly by A1 than A2 isolates, and in the former tended to be of an even size and were frequently single, whereas in A2 isolates the size was extremely variable and production was usually in clusters. Both compatibility types showed similar temperature-growth optima, but the mean growth rate of A1 isolates, over the range 15-30°C, was less than that of A2 isolates. Both types responded similarly to high temperature treatments and to a range of osmotic potentials and both showed similar pathogenicity to lupin and Pinus radiata seedlings. While both compatibility types showed similar reactions to a number of inhibitors, the growth of A2 isolates was inhibited more by rose bengal and pyronin G than that of A1 isolates. Thus, although significant differences were recorded, there was a general similarity of behaviour of isolates of both compatibility types and there were no striking differences from the recorded behaviour of the fungus in other countries.

The Relation of the Carbon-Nitrogen Ratio in the Basal Medium to Sexual Reproduction in Species of Phytophthora

Following the discovery by Haskins et al. (1964) that 3-fi-hydroxy sterols are required for the formation of oospores by Pythium species, these compounds were also found to be required for oospore formation by many species and isolates of Phytophthora (Elliott et al, 1964, 1966; Harnish and Merz, 1964; Hendrix, 1964, 1965; Hunter et al., 1965; and Leal et al., 1964). Sterol concentrations of 4-30 mg/liter have been effective. Some species and isolates formed oospores readily on synthetic media to which agar and a suitable sterol were added. Others produced oospores only on certain natural media, and still others produced few or no oospores in the laboratory. Gallegly and Lilly (1964) reported that pairings of A1 and A2 compatibility types of Phytophthora cinnamomi Rands and P. cryptogea Pethyb. & Laff. consistently produced oospores in abundance on hemp seed agar, but not on lima bean agar. Hunter et al. (1965) found that the following species produced no oospores on a medium containing 2 g/liter of D-glucose, 1 g/liter of L-asparagine, and 4 mg/liter of ,/sitosterol: P. hibernalis Carne, P. syringae (Kleb.) Kleb., P. cinnamomi, and two pairings of P. cryptogea. Some isolates of P. citricola Saw., P. ilicis Buddenhagen & Young, and P. megasperma Drechs. produced few or no oospores on the above medium. The influence of the composition of the basal medium (other than the presence of sterol) on the production of oospores by species of Phytophthora has not been studied thoroughly. The concentrations (g/liter) of the carbon and nitrogen sources used by the above investigators have ranged from 2-30 and 1.0-2.5, respectively, to give carbon to nitrogen ratios (C/N) ranging from 3.8:1 to 27.4:1. The major sources of carbon were glucose and sucrose, and of nitrogen were L-asparagine and sodium nitrate.