Oospore Formation by Phytophthora infestans in Potato Tubers

ABSTRACT The ability of Phytophthora infestans, the causal agent of potato and tomato late blight, to produce oospores in potato tuber tissue was studied in the field and under laboratory conditions. In 1998 and 2000 field experiments, the canopy of potato cvs. Alpha and Mondial, respectively, were coinoculated with A1 + A2 sporangia of the fungus, and the infected tubers collected at harvest were examined for the presence of oospores. In 1998, only 2 of 90 infected tubers had oospores, whereas none of the 90 tubers examined in 2000 had any oospores. In the latter experiment, infected tubers kept in storage up to 12 weeks after harvest had no oospores. Artificial co-inoculations of whole tubers with A1 + A2 sporangia resulted only rarely in the formation of oospores inside the tubers. Co-inoculations of potato tuber discs taken from dormant tubers 0 to 16 weeks after harvest failed to support any oospore production, whereas discs taken from sprouting tubers of >/=18 weeks after harvest allowed oospores to form. Tuber discs showed enhanced oospore formation when treated before inoculation with either sugars, amino acids, casein hydrolysate, beta-sitosterol, or chloroethylphosphonic acid. In contrast, reducing airflow into the petri dishes where potato tuber discs were incubated reduced the number of oospores produced. The number of oospores produced in tuber tissue was lower compared with that in leaf tissue regardless of the origin of isolates used. The data show that the ability of Phytophthora infestans to produce oospores in potato tuber tissue is very limited and increases with tuber aging.     

Production, survival and infectivity of oospores of Phytophthora infestans

The formation of oospores of Phytophthora infestans was studied in tomato and potato crops and volunteer plants under field conditions, and in laboratory tests with leaf discs of potato cultivars differing in their level of race-nonspecific resistance. Oospores were readily detected in blight-affected tomato leaflets and fruits, and in leaflets of field crops and volunteer potato plants. Oospores extracted from blighted potato leaflets yielded 13 oospore-derived progeny. Oospores were also produced following inoculation of leaf discs of eight potato cultivars expressing different levels of race-nonspecific resistance with a mixture of sporangia of A1 and A2 isolates. The highest numbers of oospores were produced in cvs Bintje (susceptible) and Pimpernel (resistant), and the lowest in Nicola (intermediate resistance). The relationship between lesions per leaflet and oospore incidence, affected by varying A1 : A2 ratios, was explored using a simple mathematical model, and validated by comparing actual oospore production in leaflets with multiple lesions of the race-nonspecific-resistant potato clone Lan 22-21 with the predictions generated by the model. Survival of oospores was investigated after their incorporation in either a sandy or a light clay soil in buried clay pots exposed to the local weather conditions. Over 6 years these soils were regularly assessed for their infection potential using floating leaflets in a spore-baiting bioassay. Sandy and clay soils contaminated with oospores remained infectious for 48 and 34 months, respectively, when flooded. Infections of floating potato leaflets occurred within 84–92 h and ceased after 11 days. Soil samples remained infective if dried and re-flooded on two, but not more, occasions.     

Sprinkling irrigation enhances production of oospores of phytophthora infestans in field-grown crops of potato

ABSTRACT Two field experiments were conducted to study the effect of overhead sprinkling irrigation on oospore formation by the late blight fungus Phytophthora infestans in potato. Total rain (natural + sprinkling) accumulated in treatments of experiment 1 (winter 1997 to 1998) were 765, 287, and 219 mm and treatments of experiment 2 (winter 1999 to 2000) were 641, 193, and 129 mm. Sporangia from 11 isolates of P. infestans were combined in eight pairs, seven of A1 and A2 and one of A2 and A2 mating type, and were sprayed on field-grown potato crops (42 plants per plot at 7 m(2) each) and examined for their ability to form oospores in the host tissues. In experiment 1, oospores were recorded in a total of 132 of 1,680 leaflets (7.9%), 24 of 105 stems, and 2 of 90 tubers. In experiment 2, oospores were recorded in 40 of 519 leaflets (7.7%), but not in any of the 90 stems or the 45 tubers examined. Both the proportion of leaflets containing oospores and the number of oospores per leaflet increased with time after inoculation and were dependent on the rain regime, the position of leaves on the plant, and the isolate pair combination. In both field trials, increasing the rainfall significantly enhanced oospore production in leaves. Leaf samples collected from the soil surface had significantly more oospores than those collected from the midcanopy. Two pairs in experiment 1 were more fertile than the others, whereas the pair used in experiment 2 was the least fertile. The total number of oospores per leaflet usually ranged from 10 to 100 in experiment 1, but only from 2 to 10 in experiment 2. Maximal oospore counts in the field were 200 and 50 in experiments 1 and 2, respectively, but ranged from approximately 2,000 to 12,000 oospores per leaflet in detached leaves in the laboratory. We concluded that P. infestans can produce oospores in the foliage of field-grown potato crops, especially when kept wet by regular overhead sprinkling irrigation, but production was far below that in the laboratory.     

Development of a species-specific and sensitive detection assay for Phytophthora infestans and its application for monitoring of inoculum in tubers and soil

A specific and sensitive PCR assay for the detection of Phytophthora infestans , the cause of late blight of potato, in soil and plant tissues was developed. A P. infestans -specific primer pair (INF FW2 and INF REV) was designed by comparing the aligned sequences of rDNA internal transcribed spacer regions of most of the known Phytophthora species. PCR amplification of P. infestans DNA with primers INF FW2 and INF REV generated a 613 bp product, and species specificity was demonstrated against DNA from nine other Phytophthora species and seven potato-blemish pathogens. In a single-round PCR assay, 0·5 pg pure P. infestans DNA was detectable. Sensitivity was increased to 5 fg DNA in a nested PCR assay using Peronsporales-specific-primers in the first round. As few as two sporangia or four zoospores of P. infestans could be detected using the nested assay. Procedures are described for detection of P. infestans in leaves, stem and seed potato tubers before expression of symptoms. A soil assay in which 10 oospores per 0·5 g soil were detectable was developed and validated using samples of field soil. The PCR assay was used to examine the long-term survival of sexual (oospores) and asexual (sporangia and mycelium) inoculum of P. infestans in leaf material buried in a replicated experiment under natural field conditions. Oospores were consistently detected using the PCR assay up to 24 months (total length of the study) after burial in soil, whereas the sporangial inoculum was detected for only 12 months after burial. Sporangial inoculum was shown to be nonviable using a baiting assay, whereas leaf material containing oospores remained viable up to 24 months after burial.     

Formation and survival of oospores of Phytophthora infestans under natural conditions

Phytophthora infestans is able to produce oospores in leaves of potato and tomato plants after inoculation with a mixture of Al and A2 mating-type isolates. Various conditions for oospore formation were analysed. Under controlled conditions, oospores were produced in potato leaves at temperatures ranging from 5 to 25° C. In leaves of potato cultivar Bintje incubated at 15°C, oogonia and antheridia were observed 6 days after inoculation and thick-walled oospores appeared 3-4 days later. In field experiments oospores were found in leaves and stems of potato cultivars Bintje, Irene and Pimpernel and in leaves, stems and fruits of tomato cultivar Moneymaker within 2 weeks after inoculation. A bioassay was developed to test the survival of oospores in soil under various conditions. To determine whether late-blight infections derived from infectious soil were caused by oospwres, DNA fingerprinting was performed. DNA fingerprint probe RG-57 was suitable for distinguishing asexual progeny from recombinant progeny arising from soil-borne oospores. We demonstrated survival of viable, infectious oospores of P. infestans in soil during the winter of 1992–93. Oospores were not infectious from soil exposed to temperatures of 40°C or higher but in the range 35°C to as low as – 80°C for 48 h, oospores survived.     

谷子白发病抗病性研究——Ⅰ、抗病性鉴定技术的研究

 为提高谷子白发病接种效率和鉴定结果的准确性,研究了卵孢子菌土种子包衣接种鉴定技术和孢子囊(无性孢子)浸芽接种鉴定技术:(1)利用16%甲基纤维和50%京二BA剂水溶液作粘着剂,将菌:±=1:5的菌土均匀粘着在种子表面。其接种效率和鉴定准确性大大优于菌土覆盖播种层的传统鉴定方法;(2)利用孢子囊菌悬液浸泡2-3毫米的谷芽,可以造成系统侵染并可作为谷子白发病抗病性鉴定接种方法。作者还研究了孢子囊诱发和病叶保存作孢子囊源的适宜条件。     

Occurrence of the A2 mating type and oospores ofPhytophthora Infestans in potato crops in Israel

Seventeen metalaxyl-sensitive and 21 metalaxyl-resistant isolates ofPhytophthora infestans collected from blighted potato fields during the years 1983–1988 were tested for mating type on rye seed agar medium. All isolates except one (MS3, collected in 1986 at Sufa, in the western Negev) were found to belong to the A2 mating type. A2 isolates produced oospores within 2 weeks when cultured together with isolate 163 (A₁ from the U.S.A.) or with the A₁ isolate MS3 from Israel. When cultured singly, A₂ isolates produced some oospores within 4–8 weeks. Blighted potato tubers harvested from potato crops artificially inoculated with a mixture of A1+A2 sporangia were found to contain some oospores. No oospores were detected in blighted tubers harvested from A₂ + A₂ inoculated crops. It was concluded that the A2 mating type ofP. infestans has occurred in Israel since 1983 or even earlier. The rare occurrence of the A1 mating type was unexpected and indicated that sexual reproduction of the fungus in the country might be limited.     

Increased susceptibility toPseudoperonospora cubensis in virus-infectedCucurbita pepo genotypes

Pseudoperonospora cubensis developed more and larger lesions, infected greater leaf areas and produced more sporangia on virus-infected (Vi) than on virus-free (Vf) leaves ofCucurbita pepo genotypes.Pseudoperonospora cubensis from Vi and Vf leaves did not vary in sporangial dimensions (length x width) or pathogenicity to different cucurbits. The preferential development and sporulation ofP. cubensis in Vi than in Vf leaves was due to increased host susceptibility.     

Primary Infection, Lesion Productivity, and Survival of Sporangia in the Grapevine Downy Mildew Pathogen Plasmopara viticola

ABSTRACT Several aspects of grapevine downy mildew epidemiology that are fundamental to model predictions were investigated. Simple rainfall-, temperature-, and phenology-based thresholds (rain > 2.5 mm; temperature > 11 degrees C; and phenology > Eichorn and Lorenz [E&L] growth stage 12) were evaluated to forecast primary (oosporic) infection by Plasmopara viticola. The threshold was consistent across 15 years of historical data on the highly susceptible cv. Chancellor at one site, and successfully predicted the initial outbreak of downy mildew for 2 of 3 years at three additional sites. Field inoculations demonstrated that shoot tissue was susceptible to infection as early as E&L stage 5, suggesting that initial germination of oospores, rather than acquisition of host susceptibility, was probably the limiting factor in the initiation of disease outbreaks. We also found that oospores may continue to germinate and cause infections throughout the growing season, in contrast to the widely-held assumption that the supply of oospores is depleted shortly after bloom. Lesion productivity (sporangia/lesion) did not decline with age of a lesion in the absence of suitable weather to induce sporulation. However, the productivity of all lesions declined rapidly through repeated cycles of sporulation. Extremely high temperatures (i.e., one day reaching 42.8 degrees C) had an eradicative effect under vineyard conditions, and permanently reduced sporulation from existing (but not incubating) lesions to trace levels, despite a later return to weather conducive to sporulation. In fair weather, most sporangia died sometime during the daylight period immediately following their production. However, over 50% of sporangia still released zoospores after 12 to 24 h of exposure to overcast conditions.     

Differential Activity of Carboxylic Acid Amide Fungicides Against Various Developmental Stages of Phytophthora infestans

ABSTRACT Three carboxylic acid amide (CAA) fungicides, mandipropamid (MPD), dimethomorph (DMM) and iprovalicarb (IPRO) were examined for their effects on various asexual developmental stages of Phytophthora infestans in vitro and in planta. Germination of cystospores and direct germination of sporangia were inhibited with nanomole concentrations of MPD (0.005 mug/ml) and micromole concentrations of DMM (0.05 mug/ml) or IPRO (0.5 mug/ml). A temporary exposure of 1 h to CAAs was not detrimental to germination and infectivity of sporangia or cystospores. CAAs applied to cystospores at 1 h after the onset of germination did not prevent the emergence of germ tubes, but inhibited their further growth and deformed their shape. None of the fungicides affected discharge of zoospores from sporangia or the encystment (cell wall formation/assembly) of the zoospores. Mycelium growth in solid or liquid media was inhibited with micromole concentrations. CAAs mixed with sporangia and drop inoculated onto detached leaves strongly suppressed infection. Curative application at 1 day postinoculation (dpi) required higher concentrations of CAAs than preventive application to inhibit infection and lost its effectiveness at 2 dpi. When sprayed on established late blight lesions 4 days after inoculation, CAAs reduced sporangial production in a dose-dependent manner. Trans-laminar protection of potato or tomato leaves, although achieved with higher doses, was more effective with MPD than with DMM or IPRO. Shade house studies demonstrated superior control of late blight epidemics by MPD compared with the other molecules. The data suggest that germ tube formation by cystospores or sporangia is the most sensitive stage in the life cycle of P. infestans to CAAs. Of the three CAAs, MPD had the highest intrinsic activity against spore germination. This property, together with its better trans-laminar activity, makes MPD more effective than DMM or IPRO in controlling epidemics caused by P. infestans.     

Commercial Fungicide Formulations Induce In Vitro Oospore Formation and Phenotypic Change in Mating Type in Phytophthora infestans

ABSTRACT A wide range of commercially formulated fungicides cause in vitro effects on mating behavior in specific isolates of Phytophthora infestans, the causal agent of late blight of potato and tomato. Four isolates of P. infestans representing each of the four common US genotypes, US-1, US-6, US-7, and US-8 and varying in their sensitivity to metalaxyl, were exposed to a variety of fungicides used to control late blight in petri dish assays at concentrations ranging from 1 to 100 mug a.i./ml. Exposure of each of these normally heterothallic single mating type isolates of P. infestans to 9 of the 11 commercial fungicide formulations tested resulted in the formation of oospores after 2 to 4 weeks. The highest numbers of oospores were formed on media amended with Ridomil 2E (metalaxyl) and Ridomil Gold EC (mefenoxam) at 0.1 to 10 mug a.i./ml, averaging as many as 471 and 450 oospores per petri dish, respectively. Several other fungicides including Maneb, Manzate (Mancozeb), Curzate (cymoxanil + mancozeb), and Acrobat MZ (dimethomorph + mancozeb) also induced oospore formation, producing from 0 to 200 oospores per plate at fungicide concentrations from 0.1 to 10 mug a.i./ml. The metalaxyl resistant isolates formed oospores in response to the fungicides more often than the metalaxyl sensitive isolates. No oospores were formed on media amended with Bravo (chlorothalonil) or Tattoo C (chlorothalonil + propamocarb HCl) and these compounds completely suppressed growth of the isolates at 0.1 and 1 mug a.i./ml. Three metalaxyl resistant A2 isolates mated with both A1 and A2 isolates after exposure to the fungicides Ridomil 2E and Ridomil Gold EC. Alterations in mating type expression were also observed in a metalaxyl sensitive A1 isolate after exposure to Benlate (benomyl). Copious amounts of chemicals are applied annually to potato and tomato production areas to control late blight. Our results indicate that a wide range of chemically diverse fungicides can induce normally heterothallic metalaxyl resistant isolates of P. infestans to form oospores in vitro after short exposures to the fungicides.     

恶疫霉有性杂交后代的生物学研究

 将2个带有不同抗药性标记的恶疫霉菌株配对,培养36 d后诱导其卵孢子萌发,从3760个单卵孢株中,获得50株带有双亲标记的杂交个体。对其中32株杂交个体的生长速率、有性和无性繁殖能力、卵孢子和游动孢子萌发率、致病力及对高温的耐受力等生物学性状进行测定。结果显示,测试的32株杂交个体在LBA培养基上均能较好地生长,有24株的杂交个体的生长速率介于2亲本之间;在LBA和SL培养基上,大多数杂交个体均能产生较大数量的卵孢子,有37%的杂交个体在SL培养基上产卵孢子能力显著大于双亲,仅有1个杂交个体不产生卵孢子;在人工培养条件下,大多数供试杂交个体可以产生较大数量的孢子囊,其中15株杂交个体产孢子囊能力处于双亲之间;被测的杂交个体产生的卵孢子或游动孢子均可以萌发形成有效的单孢株,有22株的杂交个体的卵孢子萌发率大于50%;被测的杂交个体接种在苹果上都有较强的致病力,有16株杂交个体的致病力显著大于双亲。表明在群体水平上恶疫霉有性杂交后代具有较强的生活能力,提示同宗配合恶疫霉不同菌株间的有性重组,对该种的群体遗传多样性可能具重要作用。     

Insights into Phytophthora ramorum sporulation: epidemiological and evolutionary implications1

Two aspects on the epidemiology of Phytophthora ramorum are discussed. Firstly, in order to elucidate its spatial scale of dispersal, its modes of asexual sporulation in culture and in planta are examined and compared to those of the also aerially dispersed P. infestans. On agar media, P. ramorum sporangia are slightly sticky, tend to aggregate in clusters, and are not shed into the air even when violently shaken or subjected to strong air humidity changes. After inoculating the underside of leaves from an assortment of Mediterranean woody species, no evidence of any adaptations for wind dispersal was observed. On some hosts, it formed sporangiomata (clusters of sporangia) on the upperside. Our observations agree with previous field research in that P. ramorum is rain‐splash rather than wind dispersed, as opposed to P. infestans. Secondly, in Petri dish cultures both species sporulate heavily. However, a steep reduction in sporangial density (sporangia cm^?2) was found on leaves of susceptible Mediterranean species for P. ramorum and a progressive one for P. infestans when this was inoculated on leaves of susceptible potato and other members of the Solanaceae. Furthermore, there was a negative correlation between sporangial density and virulence. We propose a similar pattern of a trade‐off between spore production and virulence to that observed by Thrall & Burdon (2003 ) for the Linum‐Melampsora plant‐pathogen system. Epidemiological and evolutionary implications are discussed.     

Epidemiological importance of Solanum sisymbriifolium, S. nigrum and S. dulcamara as alternative hosts for Phytophthora infestans

Lesions of Phytophthora infestans were found on woody nightshade ( Solanum dulcamara ), black nightshade ( S. nigrum ) and S. sisymbriifolium during a nationwide late blight survey in the Netherlands in 1999 and 2000. Pathogenicity and spore production of P. infestans isolates collected from potato ( S. tuberosum ), S. nigrum , S. dulcamara and S. sisymbriifolium were determined on several host plant species, and oospore formation in naturally infected and inoculated foliage of hosts was quantified. The present population of P. infestans in the Netherlands is pathogenic on S. nigrum , S. dulcamara and S. sisymbriifolium . Oospores were produced in leaves of S. nigrum , S. dulcamara and S. sisymbriifolium following infection with A1 and A2 isolates. Therefore these plant species should be regarded as alternative hosts for the late blight pathogen. In the case of S. nigrum and S. dulcamara infection was a relatively rare event, suggesting that diseased plants do not significantly contribute to the overall late blight disease pressure present in potato-production areas. Oospore production in ageing S. nigrum and S. dulcamara plants in autumn, however, may generate a considerable source of (auto) infections in following years. Considerable numbers of sporangia and oospores were produced on S. sisymbriifolium following infection with P. infestans . Additional field infection data are needed to evaluate the epidemiological consequences of a commercial introduction of S. sisymbriifolium as a potato cyst nematode trap crop.     

Environmental and Genetic Factors Influencing Self-Fertility in Phytophthora infestans

ABSTRACT Phytophthora infestans is generally regarded as heterothallic-requiring physical proximity of two individuals of different mating type (A1 and A2) for oosporogenesis. Recent reports of limited selfing in young cultures of this oomycete stimulated us to investigate factors contributing to the phenomenon. The ability to produce oospores rapidly (within 2 weeks) in pure, single individual cultures (self-fertility) was tested in 116 individual isolates. The 116 isolates were from geographically diverse locations (16 countries) and were genetically diverse. Mating type and growth medium were the most prominent factors in determining if an isolate would be self-fertile. The majority of A2 isolates (45 of 47 tested) produced oospores when grown on a 50:50 mixture of V8 and rye B medium. In contrast, the majority of A1 isolates (65 of 69 tested) did not produce oospores on this medium. None of the 116 isolates produced oospores when grown on rye B medium (with no V8 juice). Further tests on representative A1 and A2 isolates revealed that oatmeal agar, tomato juice agar, and V8-juice agar all induced the A2 mating type isolate to produce oospores but did not induce the A1 mating type isolate to produce oospores. Calcium carbonate and pH did not alter the self-fertile oospore production in either A1 or A2 mating type isolates. For in vivo tests, the application of fungicide to potato or tomato leaf tissue either before or after inoculation did not stimulate any individual isolate (one A2 and one A1 isolate) to produce oospores in infected tissue. However, in all of the controls for all experiments (in vivo and in vitro), many oospores were produced rapidly if both strains grew in physical proximity.     

Identities and pathogenicities of phytophthora spp. causing root rot of red raspberry

Phytophthora syringae, P. drechsleri, P. cactarutn, P. cambiuora and P. megasperma were isolated from the roots of red raspberry plants affected by severe root and crown rot with associated cane death. Phytophthora megasperma occurred most frequently and consisted of two types of isolates which differed in colony morphology, growth rates, and oogonial, oospore, sporangial and zoospore size, and pathogenicity to a range of plants. One type with large oospores was typical of P. megasperma var. megasperma , and was non-pathogenic to red raspberries, while the other with smaller oospores and which grew more slowly in culture than the first, was highly pathogenic, producing symptoms similar to those observed in the field. Highly pathogenic isolates from Germany and the USA were of this type.
All red and black raspberry cultivars tested were susceptible to the pathogenic type, although North American cultivars were generally less affected than British ones. Inoculated plants had reduced shoot and root weights, stem lesions and wilted and yellowed leaves. The blackberry × raspberry hybrid Tayberry and its blackberry parent were immune.
Phytophthora drechsleri, P. cactorum and P. cambivora produced small to moderate amounts of root rot on red raspberry, and P. cambivora also caused slight symptoms on shoots.     

Sugar beet extract induces defence against Phytophthora infestans in potato plants

The aim of this study was to find a natural and cheap agent that could induce defence responses in potato plants to combat Phytophthora infestans, which causes late blight disease that is one of the most devastating plant pathogens in agriculture. We tested whether a sugar beet extract (SBE), derived through a simple extraction procedure from a large-scale plant waste product, induced resistance under green-house conditions. In three potato genotypes differing in their level of resistance to P. infestans (two susceptible genotypes: Desiree and Bintje and one partially resistant: Ovatio), treatment with SBE resulted in significant reduction of the size of the infection lesions in a pattern similar to that seen with application of a known defence-inducing compound, β-aminobutyric acid (BABA). Lower sporangial production was also observed on SBE-treated leaves, but the reduction in sporangial production was more pronounced after BABA treatment. SBE had no apparent toxic effect on the hyphal growth of the pathogen or on the germination of sporangia. Instead, SBE triggered pathogenesis-related protein (PR-1 and PR-2) induction which suggests that the protection conferred by SBE could be via induced resistance. An array of phenolic metabolites was found in the SBE that may contribute to the defence response.     

Oospores associated with tomato seed may lead to seedborne transmission of<Emphasis Type="Italic">Phytophthora infestans</Emphasis>

Tomato fruits at the green mature stage were inoculated with a mixed sporangial suspension of A₁ and A₂ isolates ofPhytophthora infestans. Other fruits were inoculated with either A₁ or A₂ sporangia. Seeds were extracted from the blighted fruits and sown in soil or on agar media to test for the transmission of late blight to the emerging seedlings. Only 23 (0.09%) of approximately 25,000 seedlings developed symptoms. All blighted seedlings originated from fruits inoculated with mixed A₁ + A₂ sporangia. Isolates ofP. infestans recovered from the emerging blighted seedlings were seemingly of oosporic origin, as they differed phenotypically (mating type, virulence, sensitivity to metalaxyl) from the parent isolates used to inoculate the fruits. The results suggest that transmission ofP. infestans might occur by seeds extracted from fruits carrying oospores and less probably by seeds extracted from fruits having no oospores. http://www.phytoparasitica.org posting April 30, 2004.     

Competition between metalaxyl-sensitive and metalaxyl-resistant isolates of Phytophthora infestans in the absence of metalaxyl

Three metalaxyl-sensitive (MS, wild type) and three metalaxyl-resistant (MR) field isolates of Phytophthora infestans were compared, in the absence of metalaxyl, for non-competitive and competitive fitness on potato leaf and tuber tissues. When inoculated singly onto intact plants MR isolates produced larger lesions in leaflets than MS isolates, but no significant differences were recorded in infection frequency or sporulation capacity. When mixtures of MS and MR isolates were inoculated onto intact plants, all MR isolates exhibited a strong competitive ability: their proportion increased in the sporangial populations from 10 to 100% after eight to 10 sporulation cycles. In contrast, when mixtures were inoculated onto detached leaflets or tuber slices in a moisture-saturated atmosphere, only MR2 was a strong competitor; MR1 was a weak and MR3 a moderate competitor. The results showed that in intact plants all MR isolates were able to compete successfully with their MS partner isolates, because of the larger lesions they produced and the unlimited availability of host tissue. However, in detached tissues only MR2 was able to compete successfully with MS2, because of its higher infection and sporulation capabilities compared to MS2. The results explain the severe MR-induced late blight epidemics in potato crops in Israel.     

Development of a quantitative real‐time PCR assay for Phytophthora infestans and its applicability to leaf,tuber and soil samples

A sensitive real‐time polymerase chain reaction (PCR) assay was developed for the quantification of Phytophthora infestans, the cause of foliar and tuber late blight in potato. A primer pair (PinfTQF/PinfTQR) and a fluorogenic probe (PinfTQPR) were designed to perform a quantitative assay for the detection of P. infestans in leaves, tubers and soils. The assay was shown to be specific to P. infestans and the very closely taxonomically related non‐potato pathogen species P. mirabilis, P. phaseoli and P. ipomoea, but did not detect the potato pathogens P. erythroseptica and P. nicotianae. The assay was able to reliably detect P. infestans DNA at 100 fg per reaction and was effective in quantifying P. infestans in infected leaf tissue from 24 h after inoculation and also in infected symptomless tubers and diseased tubers. Attempts to detect oospores of P. infestans in naturally and artificially infested soil samples are described and compared with baiting tests and previous literature. It was not possible to detect oospores in soil samples due to problems with DNA extraction from the oospores themselves. However, the assay was shown to detect even very low levels of asexual inoculum (sporangia and mycelium) in soil. This work assembles all the necessary features of a quantitative P. infestans assay, which have previously been somewhat disparate: the sensitivity, specificity and quantitation are fully validated, the assay is shown to work in common applications in leaf and tuber tissue and the problems with P. infestans oospore detection are explored and tested experimentally.