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Oospores of Phytphthora infestans in soil provide an important new source of primary inoculum in Finland

Oospores of Phytphthora infestans in soil provide an important new source of primary inoculum in... AGRICULTURAL AND FOOD SCIENCE Vol. 13 (2004): 399–410. Oospores of P hytophthora infestans in soil provide an important new source of primary inoculum in Finland Ari Lehtinen and Asko Hannukkala MTT Agrifood Research Finland, Plant Production Research, Plant Protection, FI- 31600 Jokioinen, Finland, e-mail: ari.lehtinen@mtt.fi and asko.hannukkala@mtt.fi There have been numerous indications since the 1990s that oospore-derived primary infections play an in- creasing role in the epidemiology of potato late blight. The aim of this study was to verify that oospore- derived epidemics actually occur in Finland. For this purpose, 20 suspected foci of oospore-derived potato late blight were scouted in 2000–2002. All of these were located in fi elds in which late blight had been observed in at least one of the four previous years. Primary symptoms in these foci occurred always on the lowest leaves near or touching the ground. Leafl ets typically showing numerous primary infections or le- sions were in direct contact with the soil. In the former, oospores were observed after incubation. Soil samples from two experimental fi elds, marked by severe epidemics in most years during the last decade, caused infections in a bioassay. Both mating types were on every occasion present in groups of single lesion isolates collected from foci and the bioassay. Oospores survived over the winter, as shown by soil samples taken during the spring that infected potato leafl ets in the bioassay. The results presented indicate that oospore-derived epidemics occur in Finland. This paper also discusses the role of oospores in causing blight epidemics in Finland compared to more southern countries. Key words: potatoes, Solanum tuberosum, potato late blight, Phytophthora infestans, epidemiology, oospores, mating type, sexual reproduction, soil borne inoculum are named A1 and A2. Before the late 1970s, only Introduction the A1 mating type of P. infestans was present out- side Mexico. In the early 1980s, a new potato late Potato late blight, which is caused by the oomyc- blight population migrated from Mexico to Europe ete Phytophthora infestans (Mont.) de Bary, is a and replaced the old asexual population (Fry and worldwide devastating disease of potatoes. The Goodwin 1997). The fi rst indication of the change pathogen is heterothallic, and its two mating types was the appearance of the A2 mating type of P. in- © Agricultural and Food Science Manuscript received November 2003 399 AGRICULTURAL AND FOOD SCIENCE Lehtinen, A. & Hannukkala, A. Oospore-derived potato late blight festans in Europe (Hohl and Iselin 1984). The pres- land. Circumstantial indications of suspected ence of both mating types in a population enables the oospore-derived epidemics were gathered during sexual reproduction of P. infestans, resulting in the late blight surveys in the 1990s (Drenth et al. 1995, formation of oospores (Gallegly 1968). Monitoring Andersson et al. 1998, Zwankhuizen and Zadoks of the occurrence of the A2 mating type in Finland 2002). In the present study, an epidemic was de- was started in 1992 when it proved to be present in a fi ned to be oospore derived if the following four very low proportion in the potato late blight popula- conditions were fulfi lled: tion. Unfortunately, no older isolates of the pathogen 1) Foci appearing relatively early in a potato stand are available, so the exact time of appearance of A2 in a fi eld where late blight attacks had occurred remains unsolved (Hermansen et al. 2000). in at least one of the previous four years (Turk- Asexually reproducing P. infestans is able to ensteen et al. 2000). overwinter only as mycelium in surviving tubers 2) Primary lesions emerging from leaf edges in storage or cull piles or in ones left in the soil touching the soil or from stems (Andrivon during harvest that then produce volunteer plants 1995, Fernández-Pavia et al. 2001). in the next season (Zwankhuizen and Zadoks 3) Both mating types present in the early stage of 2002). In the northern climate, the cold winters the epidemic, as each oospore in the progeny limit the survival of the asexual stage of P. in- has a chance to inherit the A1 or A2 trait from festans in infected tubers in frozen soil. Therefore, its parents (Gallegly 1968, Drenth et al. 1995, the build-up of blight epidemics due to asexual Judelson et al. 1995). sources of primary inoculum was usually delayed 4) Soil collected from the suspected oospore-de- until the end of August (Mäkelä 1966, Seppänen rived foci able to infect detached potato leaves 1971). The oospores formed in a sexually repro- in bioassay (Drenth et al. 1995). ducing population provide a new serious overwin- tering source of primary inoculum independent of the survival of the tubers during cold winters. The Material and methods oospores are known to be very tolerant of low tem- peratures (Drenth et al. 1995, Fay and Fry 1997). There is convincing evidence that oospores re- A fi eld survey sulting from sexual reproduction are formed in po- tato crops under fi eld conditions throughout North- A fi eld survey was carried out in 2000–2002 to fi nd ern Europe (Drenth et al. 1995, Andersson et al. potential oospore-derived early infections initiat- 1998). Less clear is still the relative importance of ing late blight epidemics. The monitoring concen- oospores as a source of primary inoculum. trated on experimental trial sites of MTT Agrifood Oospores have been shown to persist in soil for Research Finland in Jokioinen and of the Potato 3–4 years in the Netherlands (Turkensteen et al. Research Institute in Lammi. In addition, about 30 2000). It has also been shown that oospores can farmers producing early potatoes under plastic or infect potatoes at least under greenhouse condi- fi bre cover on the south-western coast of Finland tions (Drenth et al. 1995, Strömberg et al. 1999). were reviewed annually for the occurrence of early In Sweden, suspected oospore-derived foci have attacks of late blight. been reported in a fi eld trial where a heavy attack of potato late blight was present in the previous potato crop (Andersson et al. 1998). The presence Primary lesions and oospore formation of both mating types and oospores in infected leaf- lets in the foci supports the hypothesis that oospores When a blight focus was found, observations were were responsible for the outbreak of the epidemic. made on the development of symptoms in the low- The aim of the present study was to verify that est leaves touching the ground. Leaves with mo- oospore-derived epidemics actually occur in Fin- saic-like symptoms were collected and incubated 400 AGRICULTURAL AND FOOD SCIENCE Vol. 13 (2004): 399–410. to study the potential oospore production on them. ChemiImager 4000 (Alpha Innotech Corporation, Leaf discs 1.5 cm in diameter were cut with a cork San Leandro, CA, USA). borer and incubated for 10 to 14 days in water in Petri dishes at 15ºC with relative humidity (RH) above 90%. After incubation, the leaf discs were Mating type analysis transferred to a glass slide and fl attened under a cover glass. The occurrence of oospores of P. in- Soil samples and a number of leafl ets with a sin- festans was verifi ed using a light microscope. gle blight lesion were collected from suspected In 2000, naturally infected stems were collect- oospore-derived foci at the onset of the epidemic. ed from Jokioinen and Nummi-Pusula and exam- The rationale for the early sampling was to verify ined for the occurrence of oospores. Twenty plants whether the mating type ratio of isolates present were sampled from late blight foci from both in the early focus was in the expected range of a fi elds. The stems were cut into lengths of 5 cm in sexually reproducing population. Mating type data the laboratory and boiled in 96% ethanol for 20 from earlier surveys was analysed to compare the minutes, then bleached in 1% NaClO for 10 min- mating type ratio between suspected oospore-de- utes. Longitudinal slices of stems were mounted rived foci and air-borne epidemics. Isolates be- on glass slides with the help of a drop of lactic longing to air-borne epidemics were selected by acid, and the presence of oospores was checked including only fi elds in which at least 10 isolates with a light microscope. were collected. Surveys were carried out and iso- lates were collected and tested for mating type dur- ing the years 1994, 1997, 1998, 1999, and 2000. To determine the mating type, the leafl ets with PCR diagnostics a single lesion were incubated in Petri dishes on When primary symptoms were found on the stems, moist fi lter paper for 2–5 days in a greenhouse at the whole plant was collected and stored in a freez- 15°C at > 90% RH to enhance sporulation. Some er to verify whether the infection originated from of the isolates were transferred to fresh potato leaf- the mother tuber. Samples were prepared from the lets to increase the inoculum for the mating type mother tuber, daughter tubers, stem in the middle assay. The mating type of the isolates was deter- with symptoms, and lower leaves touching the mined after incubation by pairings with known A1 ground by forcing the sap out of plant pieces with and A2 isolates. These tester isolates were 90209 a press (Elektrowerk, Behncke & Co, Hannover, (A1) and 88055 (A2) from the Netherlands, ob- Germany). A 50-μl aliquot of the sap was used for tained from Cyanamid Forschung GmbH, Germa- DNA extraction. DNA was extracted by using a ny, and 18 (A1) and 90 (A2) provided by Björn DNeasy Plant Mini Kit (Qiagen, Hilden, Germa- Anderson and Magnus Sandström, SLU, Swedish ny). The extracted DNA (0.5 μl) was amplifi ed by University of Agricultural Sciences. Mating type a nested polymerase chain reaction (PCR) in reac- tests were done on fl oating leaf discs, which has tion conditions recommended by the manufacturer proved to be a rapid method (Hermansen et al. of Dynazyme DNA polymerase (Finnzymes, Es- 2000). Most of the isolates were also isolated as poo, Finland). The specifi c primer pairs used were pure cultures on rye agar and characterised for O8-1/O8-4 and O8-3/O8-4. The result was a 258- mating type on rye agar (Gallegly and Galindo bp DNA fragment that is part of the O8 DNA fam- 1958). ily (Judelson and Tooley 2000). The temperatures and times in PCR were as described by Judelson and Tooley (2000). The PCR products (8 μl) were Soil infectivity analysis analysed by electrophoresis through a 0.8%-agar- -1 ose gel containing 0.5 μg ml of ethidium bromide Four soil samples of 10 litres were collected in and visualised by UV transillumination using a October 2000 from a fi eld in Jokioinen, where po- 401 AGRICULTURAL AND FOOD SCIENCE Lehtinen, A. & Hannukkala, A. Oospore-derived potato late blight tatoes had been grown in continuous monoculture Results since 1990. The potato crop on this particular fi eld had been heavily infected by late blight every year The fi eld survey and primary lesions except in 1995 and 1999. The soil samples were stored in an unheated warehouse subjected to temperatures fl uctuating The fi eld survey was arranged to verify that between +5 and –20°C. The samples were moved oospore-derived epidemics actually occur in Fin- to room temperature (19–21ºC) in February 2001. land. The aim of the survey was to fi nd potato late Five soil samples were collected from the same blight foci appearing relatively early in potato fi eld on 5 May 2001 before the potato crop was stands in fi elds where late blight attacks had oc- planted, although not exactly from the same sam- curred in at least one of the preceding four years pling sites as in the previous autumn. Soil samples (Turkensteen et al. 2000). In addition, the goal of 1–2 litres in volume were also acquired from 15 was to fi nd primary lesions that emerged from leaf suspected oospore-derived blight foci in eight edges touching the soil or from stems (Andrivon commercial production fi elds located in different 1995, Fernández-Pavia et al. 2001). A total of 20 parts of Finland. The samples were stored at 8– suspected oospore-derived late blight foci were 10ºC until testing. studied in the years 2000, 2001, and 2002 (Table Prior to testing, the soil samples were made as 1). Eight of them were detected in an early stage, homogeneous as possible by mixing them by hand thus providing primary lesions for observation. The in containers 10 times larger than the sample vol- fi rst lesions appeared on the leaves near or touch- ume. The infectivity of the soil samples collected ing the soil, and they were marked by mosaic-like in autumn 2000 in Jokioinen was tested in bio- discolorations indicating numerous simultaneous assays carried out from February to June 2001. infection sites per leaf (Fig. 1), or they originated The remaining soil samples were tested from Janu- from the edge of the leafl et in contact with the soil ary to March 2002. (Fig. 2). Sporulation was very weak in the leaves The spore baiting bioassay was carried out with mosaic-like symptoms and almost invisible following a modifi cation of the procedure de- without magnifi cation. The leaves died within 2–4 scribed by Drenth et al. (1995). Subsamples of days. one litre in size were placed in plastic containers In Lammi, the initial blight symptoms appeared (37 × 28 × 11 cm). The soil layer was covered in 2001 on three neighbouring stems in one row. with tap water up to a height of at most 1 cm. Secondary lesions on leaves were observed on After the water was added, samples were kept plants around the stem-infected plants. Tuber- overnight in a freezer. The next day, the samples borne inoculum was excluded since no late blight were transferred from the freezer to a greenhouse. symptoms were observed on mother tubers and The containers were incubated under a transpar- since the tubers did not contain detectable levels of ent plastic lid at 15°C in natural daylight. One P. infestans DNA (Fig. 3). It is also unlikely that day later, leafl ets were placed abaxial side up on only three neighbouring plants would have been the water. As a consequence of the amount of wa- infected by air-borne inoculum. Thus, regarding ter used, only a very small proportion of the leaf- primary symptom development, all eight described lets were in direct contact with the soil. Except at foci could be considered as oospore-derived foci. weekends, leafl ets were checked daily for symp- toms. Leafl ets with sporulating lesions were im- mediately removed from the containers, and the Formation of oospores in the fi eld sporangia were verifi ed by microscope as P. in- festans. After two weeks of incubation, symptom- A prerequisite of oospore-derived infections is the less leafl ets were removed and replaced by new formation of oospores in potato fi elds. Therefore, leafl ets. leafl ets showing multiple lesions were collected 402 AGRICULTURAL AND FOOD SCIENCE Vol. 13 (2004): 399–410. Table 1. General information, observations, and analyses made at suspected oospore-derived late blight foci investigated during the fi eld survey. 1) 1) Focus Field Municipality Altitude Latitude Site type Year Previous Type of primary symptoms Mating Oospore PCR diagnos- Leaf no. no. potato crop type assay tics of tubers infections ratio and stems in soil bioassay 1 1 Jokioinen 60°51’ 23°26’ trial 2000 since 1990 mosaic-like discoloration x leaves + Yes stem 2 1 Jokioinen trial 2001 since 1990 mosaic-like discoloration x Yes 3 1 Jokioinen trial 2002 since 1990 mosaic-like discoloration No 4 2 Nummi-Pusula 60°41’ 22°11’ garden 2000 since 1990 x leaves + No stem 5 3 Tammela 60°46’ 23°51’ trial 2000 1998 x 6 4 Ruukki 64°35’ 25°09’ trial 2000 1999 mosaic-like discoloration leaves No 7 4 Ruukki trial 2001 2000 mosaic-like discoloration leaves No 8 5 Lammi 61°05’ 25°03’ trial 2000 1996 mosaic-like discoloration x leaves 9 5 Lammi trial 2001 2000 stem blight x No 10 5 Lammi trial 2001 2000 No 11 6 Hattula 61°04’ 24°18’ garden 2001 since 1990 mosaic-like discoloration leaves 2) 12 7 Joroinen 62°11’ 27°49’ garden 2001 since1991 xYes 13 8 Merimasku 60°27’ 21°51’ fi eld 2001 2000 No 14 8 Merimasku fi eld 2001 2000 No 15 8 Merimasku fi eld 2001 2000 No 16 8 Merimasku fi eld 2001 2000 No 17 9 Kalajoki 64°11’ 24°01’ fi eld 2001 2000 No 18 9 Kalajoki fi eld 2001 2000 No 19 9 Kalajoki fi eld 2001 2000 No 20 10 Siikajoki 64°47’ 24°48’ fi eld 2001 since 1999 No 1) Measured at the geographical center of the town area 2) Not in 1999 AGRICULTURAL AND FOOD SCIENCE Lehtinen, A. & Hannukkala, A. Oospore-derived potato late blight Fig. 2. A lesion starting from the edge of a leafl et in direct Fig. 1. Numerous small mosaic-like lesions caused by P. contact with the soil. infestans in a single leaf. Fig. 3. Gel electrophoresis of amplifi cation products after polymerase chain reaction, with DNA extracted from different parts of the three potato plants showing stem blight symptoms. St: 100 bp DNA ladder; lanes 1–4: plant 1; lanes 5–8: plant 2; lanes 9–12: plant 3: lanes 1, 5, and 9: lower leaf; lanes 2, 6, and 10: stem from middle of the plant; lanes 3, 7, and 11: seed po- tato; lanes 4, 8, and 12: daughter tuber; nc: negative control. from six suspected oospore-derived foci and stems Mating type ratio and oospores in foci from the foci at Jokioinen and Nummi-Pusula (Ta- ble 1). Leaf discs derived from all of the six foci Oospores occur in batches in the soil since a single formed oospores in Petri dishes after incubation leafl et can contain thousands of oospores (Drenth (Fig. 4). Oospores were also observed in the stems et al. 1995). Thus, it is probable that multiple collected from Jokioinen, but not in those from oospores will germinate under favourable condi- Nummi-Pusula. Since the stems were analysed tions producing a mixture of A1 and A2 zoospores, right after sampling, the formation of oospores is which result in the formation of oospores in foci. possible under favourable weather conditions in The presence of both mating types in suspected Finnish potato fi elds. oospore-derived foci and air-borne epidemics was 404 AGRICULTURAL AND FOOD SCIENCE Vol. 13 (2004): 399–410. Fig. 6. Mating types of isolates from four soil samples Fig. 4. Oospores of P. infestans inside a potato leaf disc collected in Jokioinen in autumn 2000 (autumn 1–4) and detected after incubation in the laboratory in different lo- from one in spring 2001 (spring 1). Number of isolates cations and years. studied indicated within bars. studied to test if the mating type ratio or the pres- Oospore occurrence in soil ence of the oospores can support the hypothesis of the foci being oospore-derived. Soil samples were collected from suspected Both mating types were always present in pop- oospore-derived disease foci to gather indirect evi- ulations of single lesion isolates collected from dence for oospore-derived infections. Only three suspected oospore-derived foci or from bioassay soil samples taken from the 16 distinct foci were (Fig. 5 and 6). In addition, every sampled focus infective in the bioassay. The fi rst symptoms were having primary infected leafl ets and showing mul- observed 5–26 days after starting a test. In Jo kioi- tiple lesions produced oospores in leaf discs de- nen, soil samples collected from the same fi eld rived from these leafs, which indicates the pres- in both autumn 2000 and spring 2001 resulted in ence of both mating types (Fig. 4). In contrast, sporulating late blight lesions in the bioassay (Ta- 37% of fi elds with air-borne epidemics had only ble 1), indicating that oospores are able to survive one mating type present, and 37% of the remaining in the soil to the next growing season. However, fi elds had a frequency of at least 80% of the domi- despite careful mixing of the soil samples, con- nating mating type (Fig. 7). Thus, the occurrence siderable variation in the amount of infection was of both mating types was substantially more com- observed between subsamples (data not shown). mon in suspected oospore-derived populations. Thus, the bioassay and our sampling scheme may not have been sensitive enough to detect an inocu- lum capable of causing infections. Discussion It is very diffi cult to prove directly that oospores caused an infection observed in the fi eld. Therefore, we decided to investigate if the prerequisites for oospore-derived infections are fulfi lled in Finland. In addition, suspected oospore-derived late blight Fig. 5. Mating types of isolates collected from different foci were investigated to verify if the symptoms, suspected oospore-derived foci. Number of isolates stud- mating type ratio, and the presence of oospores in ied indicated within bars. 405 AGRICULTURAL AND FOOD SCIENCE Lehtinen, A. & Hannukkala, A. Oospore-derived potato late blight Fig. 7. Distribution of mating types in air-borne epidemics observed at different locations during the years 1994, 1997, 1998, 1999, and 2000. Number of isolates studied indicated within bars. 406 AGRICULTURAL AND FOOD SCIENCE Vol. 13 (2004): 399–410. soil support the hypothesis of an oospore-derived splashed by rain onto leafl ets, where they germi- infection. There is also a discussion of the role of nate, produce zoospores by indirect germination of oospores in causing blight epidemics in Finland sporangia, and infect the leafl ets. Numerous infec- compared to more southern countries. tions in a single leafl et could be explained by nu- merous zoospores having been released on the leafl et. Alternatively, zoospores could have already been released in the soil and have actively swum Prerequisites for oospore-derived onto the leafl ets touching the soil, or they could infections have been splashed onto the leafl ets by rain. In Lammi, the fi rst symptoms were discovered Prerequisites for oospore-derived infections are on the stems of three plants. Tuber-borne inoculum that oospores be formed in potato fi elds and that was excluded since no late blight symptoms were they survive until the next growing season in the observed on mother tubers, and the tubers did not soil under natural weather conditions. It has al- contain detectable levels of P. infestans DNA. It is ready been shown by Hermansen et al. (2000) also very unlikely that these stem infections could that both mating types are present in Finland. Af- have been related to an air-borne inoculum. ter incubation in moist conditions, oospores were Oospores may have stuck to the stem bases be- observed in leaves from all studied foci, and they tween the petiole and the stem when the plant was were observed even without incubation in stems emerging, or oospores or zoospores were splashed collected from Jokioinen. Thus, P. infestans can onto leaf bases by rain. Thus the stem symptoms produce oospores in Finnish potato fi elds. Soil originated most likely from oospores. samples taken in May 2001 from the same fi eld It is confusing that we did not observe lesions in Jokioinen showed that oospores survive in the on stem bases, which are typical for oospore-de- soil until the next growing season. Oospores have rived infections in Mexico and India (Fernández- also been shown to overwinter in the Netherlands Pavia et al. 2004, Singh et al. 2004). Perhaps the (Drenth et al. 1995, Turkensteen et al. 2000) and soil temperature during the early stage of potato Canada (Medina and Platt 1999). development is too low in Finland, usually around 10ºC, limiting or reducing oospore germination. Later on, an underground stem may be too well protected against infections. In our inoculation Primary lesions tests, lower stem parts of tuber bearing plants were The fi rst lesions were typically observed on the immune against P. infestans, but the plant tops lowest leaves as mosaic-like discolorations or le- were susceptible (data not shown). sions in direct contact with the soil. Oospores are an obvious explanation for lesions starting from leaf edges touching the soil. It is also diffi cult to Mating type ratio explain how numerous primary lesions on leaves in close or direct contact with the soil could be In theory, oospores originating from different initiated by an air-borne inoculum since the upper parents produce progeny possessing both mating leaves in the crop were symptomless. types in a ratio of approximately 1:1, but ratios of Germinating oospores produce germ tubes even 1:2 have been reported (Gallegly 1968, Ju- with terminal sporangia that can germinate directly delson et al. 1995). The reason for the deviation in or indirectly as asexual sporangia (Smoot et al. the mating type ratio is unknown. 1958, Erwin and Ribeiro 1996, p. 53). Since the In our study, single-lesion isolates collected oospores in the soil occur in patches where leafl ets from initial foci and from the bioassay included containing oospores have decomposed (Drenth et both mating types on every occasion. The presence al. 1995), it is plausible that some oospores are of both mating types explains the abundant forma- 407 AGRICULTURAL AND FOOD SCIENCE Lehtinen, A. & Hannukkala, A. Oospore-derived potato late blight tion of oospores in primary infected leafl ets. In Oospore infl uence on blight epidemics contrast, only one mating type was detected in 37% of the assumed air-borne epidemics. The fre- It is reasonable to expect that the emergence of a quency of the dominating mating type was at least new source of inoculum in the form of oospores 80% in three quarters of the epidemics. Thus, mat- will have an infl uence on late blight epidemics. ing type frequencies in air-borne and suspected This is particularly true in Finland, where potatoes oospore-derived populations clearly differ of each are grown in continuous monoculture or in crop other, latter being closer to a ratio of 1:1. sequences of 3–4 consecutive years of potatoes with a one-year cereal break. In addition, the cold northern climate is less favourable to the survival of asexual forms of primary inoculum than for Oospore occurrence in soil example in the Netherlands, where overwintering It was assumed that soil collected from suspected tubers have been shown to provide the main source oospore-derived foci would be able to cause infec- of primary inoculum (Zwankhuizen et al. 2000). tions in bioassay too. However, the only infectious Not surprisingly, Finnish farmers have experi- soils were collected from Jokioinen and Joroinen, enced that the control of late blight is now more where potatoes had been grown in monoculture diffi cult, and sales of fungicides against potato late without fungicide applications for the last decade. blight have as a consequence increased 3–4-fold Infections in bioassay were obviously a conse- since the 1980s (Hannukkala et al. 2003). How- quence of the long history of severe epidemics on ever, the roles of the observed change in the blight these fi elds, which enabled the accumulation of population (Fry and Goodwin 1997, Flier and oospores in the soil. Other soil samples from fi elds Turkensteen 1999) and of a possible shift towards with conventional chemical blight control were a weather pattern more conducive to an early start not infectious in the bioassay. This, together with of late blight epidemics cannot be excluded. the probably patchy occurrence of oospores in the Further studies are necessary to evaluate the soil, suggests that the accuracy of the bioassay and relative importance of oospore-derived inoculum sampling scheme was not sensitive enough to de- as a source of primary infections in Nordic coun- tect an oospore inoculum capable of causing infec- tries. In addition, the longevity of the oospores and tions. their impact on potato monoculture needs to be as- According to our data, it can be concluded that sessed. It is essential to understand the weather a suspected oospore-derived blight focus is most conditions conducive to oospore-derived epidem- likely to be oospore-derived if 1) late blight has ics in order to improve existing decision support observed in the fi eld in at least one of the four pre- systems. A more accurate timing of the fi rst fungi- vious years (Turkensteen et al. 2000), 2) a focus cide application may be an important step in im- appears before a secondary spread of late blight proving blight control in Finland. has been observed in the area, 3) primary lesions Acknowledgements. We would like to thank the Finn- are on leaf edges touching the soil or if infected ish Ministry of Agriculture and Forestry and the Finnish leaves show mosaic-like symptoms, 4) both mat- Cultural Foundation for providing fi nancial support for ing types or oospores occur in the focus (Gallegly the study. We are grateful to Dr Terhi Rantanen for per- 1968, Drenth et al. 1995, Judelson et al. 1995), and forming the PCR-diagnostics during the study, Ms Mari Helminen, Ms Marjo Segerstedt, Ms Senja Tuominen, Ms 5) soil collected from the focus can infect potato Tuija Vihervirta, and Ms Tuula Viljanen for their technical leafl ets in the bioassay (Drenth et al. 1995). How- assistance in the course of the study, as well as to Ms Anne ever, a lack of infections in the bioassay does not Rahkonen, Ms Elina Virtanen, Ms Sirkku Koskela, and Ms necessarily rule out oospores, as the resolution of Paula Ilola for providing the soil samples from the fi elds the baiting assay can be rather poor. in different parts of Finland. We also thank Prof. Aarne Kurppa and two anonymous referees for their critical read- ing of the manuscript and valuable suggestions. 408 AGRICULTURAL AND FOOD SCIENCE Vol. 13 (2004): 399–410. References Andersson, B., Sandström, M. & Strömberg, A. 1998. Indi- axyl resistance and virulence phenotype. Plant Pathol- cations of soil borne inoculum of Phytophthora in- ogy 49: 11–22. festans. Potato Research 41: 305–310. Hohl, H.R. & Iselin, K. 1984. Strains of Phytophthora in- Andrivon, D. 1995. 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Phytopathology Annales Agriculturae Fenniae 10: 69–108. 91: S28. Singh, B.P., Gupta, J., Roy, S. & Rana, D.K. 2004. Produc- Flier, W.G. & Turkensteen, L.J. 1999. Foliar aggressive- tion of Phytophthora infestans oospores in Planta and ness of Phytophthora infestans in three potato growing inoculan potential of in vitro produced oospores under regions in the Netherlands. European Journal of Plant temparate highlands and sub-tropical plains if India. Pathology 105: 381–388. Annals of Applied Biology 144: 363–370. Fry, W.E. & Goodwin, S.B. 1997. Resurgence of the Irish Smoot, J.J., Gough, F.J., Lamey, H.A., Eichenmuller, J.J. & potato famine fungus. BioScience 47: 363–371. Gallegly, M.E. 1958. Production and germination of Gallegly, M.E. 1968. Genetics of pathogenicity of Phytoph- oospores of Phytophthora infestans. Phytopathology thora infestans. Annual Review of Phytopathology 6: 48: 165–171. 375–396. Strömberg, A., Persson, L. & Wikström, M. 1999. Infection Gallegly, M.E. & Galindo, J. 1958. Mating types and of potatoes by oospores of Phytophthora infestans in oospores of Phytophthora infestans in nature in Mexi- soil. Plant Disease 83: 876. co. Phytopathology 48: 274–277. Turkensteen, L.J., Flier, W.G., Wanningen, R. & Mulder, A. Hannukkala, A., Lehtinen, A. & Rahkonen, A. 2003. Ob- 2000. Production, survival and infectivity of oospores served changes in blight epidemics and their conse- of Phytophthora infestans. Plant Pathology 49: 688– quences for blight control during the latest decade in 696. Finland. In: Schepers, H.T.A.M & Westerdijk, K. (eds.). Zwankhuizen, M.J., Govers, F. & Zadoks, J.C. 2000. Inocu- Proceedings of the seventh workshop of an European lum sources and genotypic diversity of Phytophthora network for development of an integrated control strat- infestans in Southern Flevoland, the Netherlands. Eu- egy of potato late blight. Poznan, Poland. PPO Special ropean Journal of Plant Pathology 106: 667–680. Report No 9. p. 67–72. Zwankhuizen, M.J. & Zadoks, J.C. 2002. Phytophthora in- Hermansen, A., Hannukkala, A., Nærstad, R.H. & Brurberg, festans’s 10-year truce with Holland: a long-term analy- M.B. 2000. Variation in populations of Phytophthora sis of potato late-blight epidemics in the Netherlands. infestans in Finland and Norway: mating type, metal- Plant Pathology 51: 413–423. 409 AGRICULTURAL AND FOOD SCIENCE Lehtinen, A. & Hannukkala, A. Oospore-derived potato late blight SELOSTUS Perunaruton munaitiöt maassa ovat uusi merkittävä epidemian alkulähde Suomessa Ari Lehtinen ja Asko Hannukkala MTT (Maa- ja elintarviketalouden tutkimuskeskus) Perunarutto on maailmanlaajuisesti yksi perunan tuhoi- 4. Oletetuista maatartuntapesäkkeistä otettu multanäy- simmista taudeista. Sen aiheuttaja on munasieniin kuulu- te tartuttaa perunan lehdet tarkoitukseen kehitetyssä va Phytophthora infestans (Mont.) de Bary, jolla on kaksi pyydyskasvitestissä. pariutumistyyppiä, ”sukupuolta”, A1 ja A2. Esiintyessään Maalähtöisiä ruttoepidemioita kartoitettiin 2000– toistensa läheisyydessä eri pariutumistyypin ruttokannat 2002 etsimällä aikaisin ilmaantuneita ruttopesäkkeitä pystyvät lisääntymään suvullisesti ja muodostamaan mu- tarkempia tutkimuksia varten. Ensisijaisesti toimittiin naitiöitä. A2-tyyppiä esiintyi vain Meksikossa 1980-lu- MTT:n koekentillä Jokioisissa, Perunantutkimuslaitok- vun alkuun asti. Molemmat pariutumistyypit omaava sella Lammilla sekä noin 30 varhaisperunan- ja palstavil- uusi ruttopopulaatio levisi Eurooppaan 1980-luvulla ja jelijän pelloilla, joilla otaksuttiin 1. ehdon toteutuvan. syrjäytti nopeasti vanhan populaation. Suomessa uuden Yhteensä 20 epäiltyä maatartuntapesäkettä tutkittiin ruttopopulaation edustajia havaittiin ensimmäisen kerran 2000–2002. Näissä ensimmäiset rutto-oireet ilmaantui- 1992. Koska tätä vanhempia ruttokantoja ei ole Suomesta vat alalehtiin. Normaalista poiketen alalehdet olivat saatavilla, uuden populaation tarkkaa leviämisajankohtaa yleensä ruton kirjavoittamia lukuisien tartuntojen seu- ei ole mahdollista selvittää. Vanhan populaation edustajia rauksena, ja ne tuhoutuivat muutamassa päivässä. Lähes ei kartoitusten aikana ole Suomesta havaittu. kaikissa monilaikkuisissa lehdyköissä syntyi inkuboin- Uusi ruttopopulaatio tuottaa Euroopassa suvullisen nin jälkeen runsaasti munaitiöitä, mikä osoitti molem- lisääntymisen seurauksena munaitiöitä. Munaitiöt ovat pien pariutumistyyppien läsnäolon pesäkkeessä. Suo- paksuseinäisiä ja kestäviä itiöitä, jotka säilyvät maassa raan pelloilta kerätyistä varsista löytyi myös munaitiöitä jopa 4 vuotta infektiokykyisinä. Viitteitä munaitiöiden Jokioisilta ja Nummi-Pusulasta. Koska Jokioisilta ke- aiheuttamista epidemioista on raportoitu Euroopassa väällä kerätyt multanäytteet tartuttivat perunanlehtiä Hollannista ja Ruotsista. Tämän tutkimuksen tavoittee- kasvihuoneessa tehdyssä pyydyskasvitestissä, pystyvät na oli selvittää, esiintyykö Suomessa munaitiöistä al- munaitiöt säilymään Suomessa talven yli maassa. kunsa saaneita perunaruttoepidemioita. Kirjallisuuden Munaitiöiden muodostuminen ja säilyminen maassa perusteella munaitiöistä alkava ruttoepidemia määritel- talven yli osoittaa, että Suomessa on edellytykset maa- tiin seuraavasti: tartunnalle. Ensimmäisten ruttolaikkujen ilmaantuminen 1. Ruttopesäkkeitä ilmaantuu perunakasvustoon aikai- lehdykän maata koskettavaan reunaan tai lukuisien pien- sin kesällä lohkolla, jolla on yhtenä neljästä viime ten laikkujen ilmaantuminen alalehteen on myös hel- vuodesta ollut ruttoinen perunakasvusto. pointa selittää maatartunnalla. Vielä ei kuitenkaan tiede- 2. Ensioireet ilmaantuvat lähellä maata oleviin tai sitä tä, miten yleisiä maalähtöiset epidemiat ovat. Maatar- koskettaviin lehtiin tai varsiin. tunnan hallitsemiseksi pitäisi edelleen selvittää, minkä- 3. Ruttopesäkkeessä esiintyy molempia pariutumistyyp- laiset kosteus- ja lämpöolot ovat kriittisiä tekijöitä infek- pejä edustavia kantoja ruttoepidemian alussa, koska tion etenemiselle, ja miten maatartunnan riskiä voitaisiin jälkeläisillä on suunnilleen yhtä suuri todennäköisyys nykyistä paremmin ennakoida. periä A1- tai A2-pariutumistyyppi vanhemmiltaan. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Agricultural and Food Science Unpaywall

Oospores of Phytphthora infestans in soil provide an important new source of primary inoculum in Finland

LEHTINEN, A.; HANNUKKALA, A.
Agricultural and Food Science – Dec 4, 2008
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AGRICULTURAL AND FOOD SCIENCE Vol. 13 (2004): 399–410. Oospores of P hytophthora infestans in soil provide an important new source of primary inoculum in Finland Ari Lehtinen and Asko Hannukkala MTT Agrifood Research Finland, Plant Production Research, Plant Protection, FI- 31600 Jokioinen, Finland, e-mail: ari.lehtinen@mtt.fi and asko.hannukkala@mtt.fi There have been numerous indications since the 1990s that oospore-derived primary infections play an in- creasing role in the epidemiology of potato late blight. The aim of this study was to verify that oospore- derived epidemics actually occur in Finland. For this purpose, 20 suspected foci of oospore-derived potato late blight were scouted in 2000–2002. All of these were located in fi elds in which late blight had been observed in at least one of the four previous years. Primary symptoms in these foci occurred always on the lowest leaves near or touching the ground. Leafl ets typically showing numerous primary infections or le- sions were in direct contact with the soil. In the former, oospores were observed after incubation. Soil samples from two experimental fi elds, marked by severe epidemics in most years during the last decade, caused infections in a bioassay. Both mating types were on every occasion present in groups of single lesion isolates collected from foci and the bioassay. Oospores survived over the winter, as shown by soil samples taken during the spring that infected potato leafl ets in the bioassay. The results presented indicate that oospore-derived epidemics occur in Finland. This paper also discusses the role of oospores in causing blight epidemics in Finland compared to more southern countries. Key words: potatoes, Solanum tuberosum, potato late blight, Phytophthora infestans, epidemiology, oospores, mating type, sexual reproduction, soil borne inoculum are named A1 and A2. Before the late 1970s, only Introduction the A1 mating type of P. infestans was present out- side Mexico. In the early 1980s, a new potato late Potato late blight, which is caused by the oomyc- blight population migrated from Mexico to Europe ete Phytophthora infestans (Mont.) de Bary, is a and replaced the old asexual population (Fry and worldwide devastating disease of potatoes. The Goodwin 1997). The fi rst indication of the change pathogen is heterothallic, and its two mating types was the appearance of the A2 mating type of P. in- © Agricultural and Food Science Manuscript received November 2003 399 AGRICULTURAL AND FOOD SCIENCE Lehtinen, A. & Hannukkala, A. Oospore-derived potato late blight festans in Europe (Hohl and Iselin 1984). The pres- land. Circumstantial indications of suspected ence of both mating types in a population enables the oospore-derived epidemics were gathered during sexual reproduction of P. infestans, resulting in the late blight surveys in the 1990s (Drenth et al. 1995, formation of oospores (Gallegly 1968). Monitoring Andersson et al. 1998, Zwankhuizen and Zadoks of the occurrence of the A2 mating type in Finland 2002). In the present study, an epidemic was de- was started in 1992 when it proved to be present in a fi ned to be oospore derived if the following four very low proportion in the potato late blight popula- conditions were fulfi lled: tion. Unfortunately, no older isolates of the pathogen 1) Foci appearing relatively early in a potato stand are available, so the exact time of appearance of A2 in a fi eld where late blight attacks had occurred remains unsolved (Hermansen et al. 2000). in at least one of the previous four years (Turk- Asexually reproducing P. infestans is able to ensteen et al. 2000). overwinter only as mycelium in surviving tubers 2) Primary lesions emerging from leaf edges in storage or cull piles or in ones left in the soil touching the soil or from stems (Andrivon during harvest that then produce volunteer plants 1995, Fernández-Pavia et al. 2001). in the next season (Zwankhuizen and Zadoks 3) Both mating types present in the early stage of 2002). In the northern climate, the cold winters the epidemic, as each oospore in the progeny limit the survival of the asexual stage of P. in- has a chance to inherit the A1 or A2 trait from festans in infected tubers in frozen soil. Therefore, its parents (Gallegly 1968, Drenth et al. 1995, the build-up of blight epidemics due to asexual Judelson et al. 1995). sources of primary inoculum was usually delayed 4) Soil collected from the suspected oospore-de- until the end of August (Mäkelä 1966, Seppänen rived foci able to infect detached potato leaves 1971). The oospores formed in a sexually repro- in bioassay (Drenth et al. 1995). ducing population provide a new serious overwin- tering source of primary inoculum independent of the survival of the tubers during cold winters. The Material and methods oospores are known to be very tolerant of low tem- peratures (Drenth et al. 1995, Fay and Fry 1997). There is convincing evidence that oospores re- A fi eld survey sulting from sexual reproduction are formed in po- tato crops under fi eld conditions throughout North- A fi eld survey was carried out in 2000–2002 to fi nd ern Europe (Drenth et al. 1995, Andersson et al. potential oospore-derived early infections initiat- 1998). Less clear is still the relative importance of ing late blight epidemics. The monitoring concen- oospores as a source of primary inoculum. trated on experimental trial sites of MTT Agrifood Oospores have been shown to persist in soil for Research Finland in Jokioinen and of the Potato 3–4 years in the Netherlands (Turkensteen et al. Research Institute in Lammi. In addition, about 30 2000). It has also been shown that oospores can farmers producing early potatoes under plastic or infect potatoes at least under greenhouse condi- fi bre cover on the south-western coast of Finland tions (Drenth et al. 1995, Strömberg et al. 1999). were reviewed annually for the occurrence of early In Sweden, suspected oospore-derived foci have attacks of late blight. been reported in a fi eld trial where a heavy attack of potato late blight was present in the previous potato crop (Andersson et al. 1998). The presence Primary lesions and oospore formation of both mating types and oospores in infected leaf- lets in the foci supports the hypothesis that oospores When a blight focus was found, observations were were responsible for the outbreak of the epidemic. made on the development of symptoms in the low- The aim of the present study was to verify that est leaves touching the ground. Leaves with mo- oospore-derived epidemics actually occur in Fin- saic-like symptoms were collected and incubated 400 AGRICULTURAL AND FOOD SCIENCE Vol. 13 (2004): 399–410. to study the potential oospore production on them. ChemiImager 4000 (Alpha Innotech Corporation, Leaf discs 1.5 cm in diameter were cut with a cork San Leandro, CA, USA). borer and incubated for 10 to 14 days in water in Petri dishes at 15ºC with relative humidity (RH) above 90%. After incubation, the leaf discs were Mating type analysis transferred to a glass slide and fl attened under a cover glass. The occurrence of oospores of P. in- Soil samples and a number of leafl ets with a sin- festans was verifi ed using a light microscope. gle blight lesion were collected from suspected In 2000, naturally infected stems were collect- oospore-derived foci at the onset of the epidemic. ed from Jokioinen and Nummi-Pusula and exam- The rationale for the early sampling was to verify ined for the occurrence of oospores. Twenty plants whether the mating type ratio of isolates present were sampled from late blight foci from both in the early focus was in the expected range of a fi elds. The stems were cut into lengths of 5 cm in sexually reproducing population. Mating type data the laboratory and boiled in 96% ethanol for 20 from earlier surveys was analysed to compare the minutes, then bleached in 1% NaClO for 10 min- mating type ratio between suspected oospore-de- utes. Longitudinal slices of stems were mounted rived foci and air-borne epidemics. Isolates be- on glass slides with the help of a drop of lactic longing to air-borne epidemics were selected by acid, and the presence of oospores was checked including only fi elds in which at least 10 isolates with a light microscope. were collected. Surveys were carried out and iso- lates were collected and tested for mating type dur- ing the years 1994, 1997, 1998, 1999, and 2000. To determine the mating type, the leafl ets with PCR diagnostics a single lesion were incubated in Petri dishes on When primary symptoms were found on the stems, moist fi lter paper for 2–5 days in a greenhouse at the whole plant was collected and stored in a freez- 15°C at > 90% RH to enhance sporulation. Some er to verify whether the infection originated from of the isolates were transferred to fresh potato leaf- the mother tuber. Samples were prepared from the lets to increase the inoculum for the mating type mother tuber, daughter tubers, stem in the middle assay. The mating type of the isolates was deter- with symptoms, and lower leaves touching the mined after incubation by pairings with known A1 ground by forcing the sap out of plant pieces with and A2 isolates. These tester isolates were 90209 a press (Elektrowerk, Behncke & Co, Hannover, (A1) and 88055 (A2) from the Netherlands, ob- Germany). A 50-μl aliquot of the sap was used for tained from Cyanamid Forschung GmbH, Germa- DNA extraction. DNA was extracted by using a ny, and 18 (A1) and 90 (A2) provided by Björn DNeasy Plant Mini Kit (Qiagen, Hilden, Germa- Anderson and Magnus Sandström, SLU, Swedish ny). The extracted DNA (0.5 μl) was amplifi ed by University of Agricultural Sciences. Mating type a nested polymerase chain reaction (PCR) in reac- tests were done on fl oating leaf discs, which has tion conditions recommended by the manufacturer proved to be a rapid method (Hermansen et al. of Dynazyme DNA polymerase (Finnzymes, Es- 2000). Most of the isolates were also isolated as poo, Finland). The specifi c primer pairs used were pure cultures on rye agar and characterised for O8-1/O8-4 and O8-3/O8-4. The result was a 258- mating type on rye agar (Gallegly and Galindo bp DNA fragment that is part of the O8 DNA fam- 1958). ily (Judelson and Tooley 2000). The temperatures and times in PCR were as described by Judelson and Tooley (2000). The PCR products (8 μl) were Soil infectivity analysis analysed by electrophoresis through a 0.8%-agar- -1 ose gel containing 0.5 μg ml of ethidium bromide Four soil samples of 10 litres were collected in and visualised by UV transillumination using a October 2000 from a fi eld in Jokioinen, where po- 401 AGRICULTURAL AND FOOD SCIENCE Lehtinen, A. & Hannukkala, A. Oospore-derived potato late blight tatoes had been grown in continuous monoculture Results since 1990. The potato crop on this particular fi eld had been heavily infected by late blight every year The fi eld survey and primary lesions except in 1995 and 1999. The soil samples were stored in an unheated warehouse subjected to temperatures fl uctuating The fi eld survey was arranged to verify that between +5 and –20°C. The samples were moved oospore-derived epidemics actually occur in Fin- to room temperature (19–21ºC) in February 2001. land. The aim of the survey was to fi nd potato late Five soil samples were collected from the same blight foci appearing relatively early in potato fi eld on 5 May 2001 before the potato crop was stands in fi elds where late blight attacks had oc- planted, although not exactly from the same sam- curred in at least one of the preceding four years pling sites as in the previous autumn. Soil samples (Turkensteen et al. 2000). In addition, the goal of 1–2 litres in volume were also acquired from 15 was to fi nd primary lesions that emerged from leaf suspected oospore-derived blight foci in eight edges touching the soil or from stems (Andrivon commercial production fi elds located in different 1995, Fernández-Pavia et al. 2001). A total of 20 parts of Finland. The samples were stored at 8– suspected oospore-derived late blight foci were 10ºC until testing. studied in the years 2000, 2001, and 2002 (Table Prior to testing, the soil samples were made as 1). Eight of them were detected in an early stage, homogeneous as possible by mixing them by hand thus providing primary lesions for observation. The in containers 10 times larger than the sample vol- fi rst lesions appeared on the leaves near or touch- ume. The infectivity of the soil samples collected ing the soil, and they were marked by mosaic-like in autumn 2000 in Jokioinen was tested in bio- discolorations indicating numerous simultaneous assays carried out from February to June 2001. infection sites per leaf (Fig. 1), or they originated The remaining soil samples were tested from Janu- from the edge of the leafl et in contact with the soil ary to March 2002. (Fig. 2). Sporulation was very weak in the leaves The spore baiting bioassay was carried out with mosaic-like symptoms and almost invisible following a modifi cation of the procedure de- without magnifi cation. The leaves died within 2–4 scribed by Drenth et al. (1995). Subsamples of days. one litre in size were placed in plastic containers In Lammi, the initial blight symptoms appeared (37 × 28 × 11 cm). The soil layer was covered in 2001 on three neighbouring stems in one row. with tap water up to a height of at most 1 cm. Secondary lesions on leaves were observed on After the water was added, samples were kept plants around the stem-infected plants. Tuber- overnight in a freezer. The next day, the samples borne inoculum was excluded since no late blight were transferred from the freezer to a greenhouse. symptoms were observed on mother tubers and The containers were incubated under a transpar- since the tubers did not contain detectable levels of ent plastic lid at 15°C in natural daylight. One P. infestans DNA (Fig. 3). It is also unlikely that day later, leafl ets were placed abaxial side up on only three neighbouring plants would have been the water. As a consequence of the amount of wa- infected by air-borne inoculum. Thus, regarding ter used, only a very small proportion of the leaf- primary symptom development, all eight described lets were in direct contact with the soil. Except at foci could be considered as oospore-derived foci. weekends, leafl ets were checked daily for symp- toms. Leafl ets with sporulating lesions were im- mediately removed from the containers, and the Formation of oospores in the fi eld sporangia were verifi ed by microscope as P. in- festans. After two weeks of incubation, symptom- A prerequisite of oospore-derived infections is the less leafl ets were removed and replaced by new formation of oospores in potato fi elds. Therefore, leafl ets. leafl ets showing multiple lesions were collected 402 AGRICULTURAL AND FOOD SCIENCE Vol. 13 (2004): 399–410. Table 1. General information, observations, and analyses made at suspected oospore-derived late blight foci investigated during the fi eld survey. 1) 1) Focus Field Municipality Altitude Latitude Site type Year Previous Type of primary symptoms Mating Oospore PCR diagnos- Leaf no. no. potato crop type assay tics of tubers infections ratio and stems in soil bioassay 1 1 Jokioinen 60°51’ 23°26’ trial 2000 since 1990 mosaic-like discoloration x leaves + Yes stem 2 1 Jokioinen trial 2001 since 1990 mosaic-like discoloration x Yes 3 1 Jokioinen trial 2002 since 1990 mosaic-like discoloration No 4 2 Nummi-Pusula 60°41’ 22°11’ garden 2000 since 1990 x leaves + No stem 5 3 Tammela 60°46’ 23°51’ trial 2000 1998 x 6 4 Ruukki 64°35’ 25°09’ trial 2000 1999 mosaic-like discoloration leaves No 7 4 Ruukki trial 2001 2000 mosaic-like discoloration leaves No 8 5 Lammi 61°05’ 25°03’ trial 2000 1996 mosaic-like discoloration x leaves 9 5 Lammi trial 2001 2000 stem blight x No 10 5 Lammi trial 2001 2000 No 11 6 Hattula 61°04’ 24°18’ garden 2001 since 1990 mosaic-like discoloration leaves 2) 12 7 Joroinen 62°11’ 27°49’ garden 2001 since1991 xYes 13 8 Merimasku 60°27’ 21°51’ fi eld 2001 2000 No 14 8 Merimasku fi eld 2001 2000 No 15 8 Merimasku fi eld 2001 2000 No 16 8 Merimasku fi eld 2001 2000 No 17 9 Kalajoki 64°11’ 24°01’ fi eld 2001 2000 No 18 9 Kalajoki fi eld 2001 2000 No 19 9 Kalajoki fi eld 2001 2000 No 20 10 Siikajoki 64°47’ 24°48’ fi eld 2001 since 1999 No 1) Measured at the geographical center of the town area 2) Not in 1999 AGRICULTURAL AND FOOD SCIENCE Lehtinen, A. & Hannukkala, A. Oospore-derived potato late blight Fig. 2. A lesion starting from the edge of a leafl et in direct Fig. 1. Numerous small mosaic-like lesions caused by P. contact with the soil. infestans in a single leaf. Fig. 3. Gel electrophoresis of amplifi cation products after polymerase chain reaction, with DNA extracted from different parts of the three potato plants showing stem blight symptoms. St: 100 bp DNA ladder; lanes 1–4: plant 1; lanes 5–8: plant 2; lanes 9–12: plant 3: lanes 1, 5, and 9: lower leaf; lanes 2, 6, and 10: stem from middle of the plant; lanes 3, 7, and 11: seed po- tato; lanes 4, 8, and 12: daughter tuber; nc: negative control. from six suspected oospore-derived foci and stems Mating type ratio and oospores in foci from the foci at Jokioinen and Nummi-Pusula (Ta- ble 1). Leaf discs derived from all of the six foci Oospores occur in batches in the soil since a single formed oospores in Petri dishes after incubation leafl et can contain thousands of oospores (Drenth (Fig. 4). Oospores were also observed in the stems et al. 1995). Thus, it is probable that multiple collected from Jokioinen, but not in those from oospores will germinate under favourable condi- Nummi-Pusula. Since the stems were analysed tions producing a mixture of A1 and A2 zoospores, right after sampling, the formation of oospores is which result in the formation of oospores in foci. possible under favourable weather conditions in The presence of both mating types in suspected Finnish potato fi elds. oospore-derived foci and air-borne epidemics was 404 AGRICULTURAL AND FOOD SCIENCE Vol. 13 (2004): 399–410. Fig. 6. Mating types of isolates from four soil samples Fig. 4. Oospores of P. infestans inside a potato leaf disc collected in Jokioinen in autumn 2000 (autumn 1–4) and detected after incubation in the laboratory in different lo- from one in spring 2001 (spring 1). Number of isolates cations and years. studied indicated within bars. studied to test if the mating type ratio or the pres- Oospore occurrence in soil ence of the oospores can support the hypothesis of the foci being oospore-derived. Soil samples were collected from suspected Both mating types were always present in pop- oospore-derived disease foci to gather indirect evi- ulations of single lesion isolates collected from dence for oospore-derived infections. Only three suspected oospore-derived foci or from bioassay soil samples taken from the 16 distinct foci were (Fig. 5 and 6). In addition, every sampled focus infective in the bioassay. The fi rst symptoms were having primary infected leafl ets and showing mul- observed 5–26 days after starting a test. In Jo kioi- tiple lesions produced oospores in leaf discs de- nen, soil samples collected from the same fi eld rived from these leafs, which indicates the pres- in both autumn 2000 and spring 2001 resulted in ence of both mating types (Fig. 4). In contrast, sporulating late blight lesions in the bioassay (Ta- 37% of fi elds with air-borne epidemics had only ble 1), indicating that oospores are able to survive one mating type present, and 37% of the remaining in the soil to the next growing season. However, fi elds had a frequency of at least 80% of the domi- despite careful mixing of the soil samples, con- nating mating type (Fig. 7). Thus, the occurrence siderable variation in the amount of infection was of both mating types was substantially more com- observed between subsamples (data not shown). mon in suspected oospore-derived populations. Thus, the bioassay and our sampling scheme may not have been sensitive enough to detect an inocu- lum capable of causing infections. Discussion It is very diffi cult to prove directly that oospores caused an infection observed in the fi eld. Therefore, we decided to investigate if the prerequisites for oospore-derived infections are fulfi lled in Finland. In addition, suspected oospore-derived late blight Fig. 5. Mating types of isolates collected from different foci were investigated to verify if the symptoms, suspected oospore-derived foci. Number of isolates stud- mating type ratio, and the presence of oospores in ied indicated within bars. 405 AGRICULTURAL AND FOOD SCIENCE Lehtinen, A. & Hannukkala, A. Oospore-derived potato late blight Fig. 7. Distribution of mating types in air-borne epidemics observed at different locations during the years 1994, 1997, 1998, 1999, and 2000. Number of isolates studied indicated within bars. 406 AGRICULTURAL AND FOOD SCIENCE Vol. 13 (2004): 399–410. soil support the hypothesis of an oospore-derived splashed by rain onto leafl ets, where they germi- infection. There is also a discussion of the role of nate, produce zoospores by indirect germination of oospores in causing blight epidemics in Finland sporangia, and infect the leafl ets. Numerous infec- compared to more southern countries. tions in a single leafl et could be explained by nu- merous zoospores having been released on the leafl et. Alternatively, zoospores could have already been released in the soil and have actively swum Prerequisites for oospore-derived onto the leafl ets touching the soil, or they could infections have been splashed onto the leafl ets by rain. In Lammi, the fi rst symptoms were discovered Prerequisites for oospore-derived infections are on the stems of three plants. Tuber-borne inoculum that oospores be formed in potato fi elds and that was excluded since no late blight symptoms were they survive until the next growing season in the observed on mother tubers, and the tubers did not soil under natural weather conditions. It has al- contain detectable levels of P. infestans DNA. It is ready been shown by Hermansen et al. (2000) also very unlikely that these stem infections could that both mating types are present in Finland. Af- have been related to an air-borne inoculum. ter incubation in moist conditions, oospores were Oospores may have stuck to the stem bases be- observed in leaves from all studied foci, and they tween the petiole and the stem when the plant was were observed even without incubation in stems emerging, or oospores or zoospores were splashed collected from Jokioinen. Thus, P. infestans can onto leaf bases by rain. Thus the stem symptoms produce oospores in Finnish potato fi elds. Soil originated most likely from oospores. samples taken in May 2001 from the same fi eld It is confusing that we did not observe lesions in Jokioinen showed that oospores survive in the on stem bases, which are typical for oospore-de- soil until the next growing season. Oospores have rived infections in Mexico and India (Fernández- also been shown to overwinter in the Netherlands Pavia et al. 2004, Singh et al. 2004). Perhaps the (Drenth et al. 1995, Turkensteen et al. 2000) and soil temperature during the early stage of potato Canada (Medina and Platt 1999). development is too low in Finland, usually around 10ºC, limiting or reducing oospore germination. Later on, an underground stem may be too well protected against infections. In our inoculation Primary lesions tests, lower stem parts of tuber bearing plants were The fi rst lesions were typically observed on the immune against P. infestans, but the plant tops lowest leaves as mosaic-like discolorations or le- were susceptible (data not shown). sions in direct contact with the soil. Oospores are an obvious explanation for lesions starting from leaf edges touching the soil. It is also diffi cult to Mating type ratio explain how numerous primary lesions on leaves in close or direct contact with the soil could be In theory, oospores originating from different initiated by an air-borne inoculum since the upper parents produce progeny possessing both mating leaves in the crop were symptomless. types in a ratio of approximately 1:1, but ratios of Germinating oospores produce germ tubes even 1:2 have been reported (Gallegly 1968, Ju- with terminal sporangia that can germinate directly delson et al. 1995). The reason for the deviation in or indirectly as asexual sporangia (Smoot et al. the mating type ratio is unknown. 1958, Erwin and Ribeiro 1996, p. 53). Since the In our study, single-lesion isolates collected oospores in the soil occur in patches where leafl ets from initial foci and from the bioassay included containing oospores have decomposed (Drenth et both mating types on every occasion. The presence al. 1995), it is plausible that some oospores are of both mating types explains the abundant forma- 407 AGRICULTURAL AND FOOD SCIENCE Lehtinen, A. & Hannukkala, A. Oospore-derived potato late blight tion of oospores in primary infected leafl ets. In Oospore infl uence on blight epidemics contrast, only one mating type was detected in 37% of the assumed air-borne epidemics. The fre- It is reasonable to expect that the emergence of a quency of the dominating mating type was at least new source of inoculum in the form of oospores 80% in three quarters of the epidemics. Thus, mat- will have an infl uence on late blight epidemics. ing type frequencies in air-borne and suspected This is particularly true in Finland, where potatoes oospore-derived populations clearly differ of each are grown in continuous monoculture or in crop other, latter being closer to a ratio of 1:1. sequences of 3–4 consecutive years of potatoes with a one-year cereal break. In addition, the cold northern climate is less favourable to the survival of asexual forms of primary inoculum than for Oospore occurrence in soil example in the Netherlands, where overwintering It was assumed that soil collected from suspected tubers have been shown to provide the main source oospore-derived foci would be able to cause infec- of primary inoculum (Zwankhuizen et al. 2000). tions in bioassay too. However, the only infectious Not surprisingly, Finnish farmers have experi- soils were collected from Jokioinen and Joroinen, enced that the control of late blight is now more where potatoes had been grown in monoculture diffi cult, and sales of fungicides against potato late without fungicide applications for the last decade. blight have as a consequence increased 3–4-fold Infections in bioassay were obviously a conse- since the 1980s (Hannukkala et al. 2003). How- quence of the long history of severe epidemics on ever, the roles of the observed change in the blight these fi elds, which enabled the accumulation of population (Fry and Goodwin 1997, Flier and oospores in the soil. Other soil samples from fi elds Turkensteen 1999) and of a possible shift towards with conventional chemical blight control were a weather pattern more conducive to an early start not infectious in the bioassay. This, together with of late blight epidemics cannot be excluded. the probably patchy occurrence of oospores in the Further studies are necessary to evaluate the soil, suggests that the accuracy of the bioassay and relative importance of oospore-derived inoculum sampling scheme was not sensitive enough to de- as a source of primary infections in Nordic coun- tect an oospore inoculum capable of causing infec- tries. In addition, the longevity of the oospores and tions. their impact on potato monoculture needs to be as- According to our data, it can be concluded that sessed. It is essential to understand the weather a suspected oospore-derived blight focus is most conditions conducive to oospore-derived epidem- likely to be oospore-derived if 1) late blight has ics in order to improve existing decision support observed in the fi eld in at least one of the four pre- systems. A more accurate timing of the fi rst fungi- vious years (Turkensteen et al. 2000), 2) a focus cide application may be an important step in im- appears before a secondary spread of late blight proving blight control in Finland. has been observed in the area, 3) primary lesions Acknowledgements. We would like to thank the Finn- are on leaf edges touching the soil or if infected ish Ministry of Agriculture and Forestry and the Finnish leaves show mosaic-like symptoms, 4) both mat- Cultural Foundation for providing fi nancial support for ing types or oospores occur in the focus (Gallegly the study. We are grateful to Dr Terhi Rantanen for per- 1968, Drenth et al. 1995, Judelson et al. 1995), and forming the PCR-diagnostics during the study, Ms Mari Helminen, Ms Marjo Segerstedt, Ms Senja Tuominen, Ms 5) soil collected from the focus can infect potato Tuija Vihervirta, and Ms Tuula Viljanen for their technical leafl ets in the bioassay (Drenth et al. 1995). How- assistance in the course of the study, as well as to Ms Anne ever, a lack of infections in the bioassay does not Rahkonen, Ms Elina Virtanen, Ms Sirkku Koskela, and Ms necessarily rule out oospores, as the resolution of Paula Ilola for providing the soil samples from the fi elds the baiting assay can be rather poor. in different parts of Finland. We also thank Prof. Aarne Kurppa and two anonymous referees for their critical read- ing of the manuscript and valuable suggestions. 408 AGRICULTURAL AND FOOD SCIENCE Vol. 13 (2004): 399–410. References Andersson, B., Sandström, M. & Strömberg, A. 1998. Indi- axyl resistance and virulence phenotype. Plant Pathol- cations of soil borne inoculum of Phytophthora in- ogy 49: 11–22. festans. Potato Research 41: 305–310. Hohl, H.R. & Iselin, K. 1984. Strains of Phytophthora in- Andrivon, D. 1995. 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Infection Gallegly, M.E. & Galindo, J. 1958. Mating types and of potatoes by oospores of Phytophthora infestans in oospores of Phytophthora infestans in nature in Mexi- soil. Plant Disease 83: 876. co. Phytopathology 48: 274–277. Turkensteen, L.J., Flier, W.G., Wanningen, R. & Mulder, A. Hannukkala, A., Lehtinen, A. & Rahkonen, A. 2003. Ob- 2000. Production, survival and infectivity of oospores served changes in blight epidemics and their conse- of Phytophthora infestans. Plant Pathology 49: 688– quences for blight control during the latest decade in 696. Finland. In: Schepers, H.T.A.M & Westerdijk, K. (eds.). Zwankhuizen, M.J., Govers, F. & Zadoks, J.C. 2000. Inocu- Proceedings of the seventh workshop of an European lum sources and genotypic diversity of Phytophthora network for development of an integrated control strat- infestans in Southern Flevoland, the Netherlands. Eu- egy of potato late blight. Poznan, Poland. PPO Special ropean Journal of Plant Pathology 106: 667–680. Report No 9. p. 67–72. Zwankhuizen, M.J. & Zadoks, J.C. 2002. Phytophthora in- Hermansen, A., Hannukkala, A., Nærstad, R.H. & Brurberg, festans’s 10-year truce with Holland: a long-term analy- M.B. 2000. Variation in populations of Phytophthora sis of potato late-blight epidemics in the Netherlands. infestans in Finland and Norway: mating type, metal- Plant Pathology 51: 413–423. 409 AGRICULTURAL AND FOOD SCIENCE Lehtinen, A. & Hannukkala, A. Oospore-derived potato late blight SELOSTUS Perunaruton munaitiöt maassa ovat uusi merkittävä epidemian alkulähde Suomessa Ari Lehtinen ja Asko Hannukkala MTT (Maa- ja elintarviketalouden tutkimuskeskus) Perunarutto on maailmanlaajuisesti yksi perunan tuhoi- 4. Oletetuista maatartuntapesäkkeistä otettu multanäy- simmista taudeista. Sen aiheuttaja on munasieniin kuulu- te tartuttaa perunan lehdet tarkoitukseen kehitetyssä va Phytophthora infestans (Mont.) de Bary, jolla on kaksi pyydyskasvitestissä. pariutumistyyppiä, ”sukupuolta”, A1 ja A2. Esiintyessään Maalähtöisiä ruttoepidemioita kartoitettiin 2000– toistensa läheisyydessä eri pariutumistyypin ruttokannat 2002 etsimällä aikaisin ilmaantuneita ruttopesäkkeitä pystyvät lisääntymään suvullisesti ja muodostamaan mu- tarkempia tutkimuksia varten. Ensisijaisesti toimittiin naitiöitä. A2-tyyppiä esiintyi vain Meksikossa 1980-lu- MTT:n koekentillä Jokioisissa, Perunantutkimuslaitok- vun alkuun asti. Molemmat pariutumistyypit omaava sella Lammilla sekä noin 30 varhaisperunan- ja palstavil- uusi ruttopopulaatio levisi Eurooppaan 1980-luvulla ja jelijän pelloilla, joilla otaksuttiin 1. ehdon toteutuvan. syrjäytti nopeasti vanhan populaation. Suomessa uuden Yhteensä 20 epäiltyä maatartuntapesäkettä tutkittiin ruttopopulaation edustajia havaittiin ensimmäisen kerran 2000–2002. Näissä ensimmäiset rutto-oireet ilmaantui- 1992. Koska tätä vanhempia ruttokantoja ei ole Suomesta vat alalehtiin. Normaalista poiketen alalehdet olivat saatavilla, uuden populaation tarkkaa leviämisajankohtaa yleensä ruton kirjavoittamia lukuisien tartuntojen seu- ei ole mahdollista selvittää. Vanhan populaation edustajia rauksena, ja ne tuhoutuivat muutamassa päivässä. Lähes ei kartoitusten aikana ole Suomesta havaittu. kaikissa monilaikkuisissa lehdyköissä syntyi inkuboin- Uusi ruttopopulaatio tuottaa Euroopassa suvullisen nin jälkeen runsaasti munaitiöitä, mikä osoitti molem- lisääntymisen seurauksena munaitiöitä. Munaitiöt ovat pien pariutumistyyppien läsnäolon pesäkkeessä. Suo- paksuseinäisiä ja kestäviä itiöitä, jotka säilyvät maassa raan pelloilta kerätyistä varsista löytyi myös munaitiöitä jopa 4 vuotta infektiokykyisinä. Viitteitä munaitiöiden Jokioisilta ja Nummi-Pusulasta. Koska Jokioisilta ke- aiheuttamista epidemioista on raportoitu Euroopassa väällä kerätyt multanäytteet tartuttivat perunanlehtiä Hollannista ja Ruotsista. Tämän tutkimuksen tavoittee- kasvihuoneessa tehdyssä pyydyskasvitestissä, pystyvät na oli selvittää, esiintyykö Suomessa munaitiöistä al- munaitiöt säilymään Suomessa talven yli maassa. kunsa saaneita perunaruttoepidemioita. Kirjallisuuden Munaitiöiden muodostuminen ja säilyminen maassa perusteella munaitiöistä alkava ruttoepidemia määritel- talven yli osoittaa, että Suomessa on edellytykset maa- tiin seuraavasti: tartunnalle. Ensimmäisten ruttolaikkujen ilmaantuminen 1. Ruttopesäkkeitä ilmaantuu perunakasvustoon aikai- lehdykän maata koskettavaan reunaan tai lukuisien pien- sin kesällä lohkolla, jolla on yhtenä neljästä viime ten laikkujen ilmaantuminen alalehteen on myös hel- vuodesta ollut ruttoinen perunakasvusto. pointa selittää maatartunnalla. Vielä ei kuitenkaan tiede- 2. Ensioireet ilmaantuvat lähellä maata oleviin tai sitä tä, miten yleisiä maalähtöiset epidemiat ovat. Maatar- koskettaviin lehtiin tai varsiin. tunnan hallitsemiseksi pitäisi edelleen selvittää, minkä- 3. Ruttopesäkkeessä esiintyy molempia pariutumistyyp- laiset kosteus- ja lämpöolot ovat kriittisiä tekijöitä infek- pejä edustavia kantoja ruttoepidemian alussa, koska tion etenemiselle, ja miten maatartunnan riskiä voitaisiin jälkeläisillä on suunnilleen yhtä suuri todennäköisyys nykyistä paremmin ennakoida. periä A1- tai A2-pariutumistyyppi vanhemmiltaan.

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