Pathogenicity and sporulation of Phytophthora pinifolia on Pinus radiata in Chile
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Phytophthora pinifolia causes the needle and shoot disease of Pinus radiata known as Daño Foliar del Pino (DFP) in Chile. The first pathogenicity trials with this organism utilized mycelial plugs placed on stem wounds. These resulted in lesions in the tissue, but did not reproduce the resinous bands on the needles, which are the most characteristics symptoms of the disease under natural conditions. In this study, stem inoculations were repeated, but to complete Koch’s postulates fully, and to confirm that P. pinifolia causes the symptoms observed on naturally infected trees, zoospore/sporangial suspensions were used to inoculate pine foliage. This method produced the same symptoms observed on needles infected naturally. These results confirm that P. pinifolia is the causal agent of the Daño Foliar del Pino on P. radiata in Chile and successfully completed Koch’s postulates for the first time. Pathogenicity tests on different Pinus spp. and hybrids showed a wide range of responses to inoculation with P. pinifolia mycelial plugs, from highly susceptible to resistant. Monitoring of sporulation revealed that the sporangia commonly remain on the needles for extended periods of time and their frequency of occurrence and dispersal appear to increase during the rainy season.
KeywordsDaño Foliar del Pino Phytophthora pinifolia Pinus radiata Needle disease Sporulation monitoring
Phytophthora pinifolia is a recently described pathogen that causes the disease known as Daño Foliar del Pino (DFP) on Pinus radiata in Chile (Durán et al. 2008). DFP has caused significant damage to the foliage of P. radiata growing in plantations in the coastal area of the Biobío and Los Ríos Regions (Durán et al. 2008). Symptoms of the disease were first observed on the Arauco coast (37°17′S; 73°36′W) in July 2004 on trees including a range of age classes. This was followed by mortality of one and two-year-old plantations in October 2004 (Durán et al. 2008). Annual monitoring showed a clear increase in the area affected over time, from 3,300 ha in 2004, to 30,000 ha in 2005 and 54,000 ha in 2006, with varying levels of severity across the region. The level of infection and damage decreased substantially in the subsequent years to 21,000 ha in 2007, 1,500 ha in 2008 and less than 1,000 ha between 2009 and 2011 (R. Gómez, pers. comm.).
Daño Foliar del Pino is characterized by relatively rapid death of infected needles followed by the defoliation of trees (Durán et al. 2008). Infection is normally observed from autumn to late spring, which is also the main rainfall season. Infected needles typically display distinct resinous bands on their laminas. When the infections reach the needle bases, resin exudes from the points of attachment causing death of the cambial cells surrounding the fascicles and generating small cankers at the contact point between the needles and the stem. The dead needles remain attached to the stems until the late spring when they fall from the trees and branches. While much is known about the development of the disease caused by P. pinifolia, there is little knowledge regarding the biology of the pathogen including issues such as its sporulation, dispersal and how it infects trees.
Pinus radiata plantations in Chile have in the past been affected by shoot and needle diseases caused by Dothistroma septosporum, Diplodia pinea and Cyclaneusma minus (Toro and Gessel 1999; Ahumada 2003; Barnes et al. 2004). Damage has also been caused by European pine shoot moth, Rhyacionia buoliana, which has been the most important constraint to plantation forestry in the past (Lanfranco 2000; Ahumada 2003). However, the increase of areas infested by the woodwasp, Sirex noctilio and the emergence of DFP are by far the most important problems currently facing P. radiata forestry in Chile.
Phytophthora spp. are amongst the most serious and invasive plant pathogens on both agricultural and forestry crops (Fry and Goodwin 1997; Rizzo et al. 2002). In terms of forestry, the recent emergence of species such as Phytophthora ramorum and P. austrocedrae (Rizzo et al. 2002; Greslebin et al. 2007) has reinforced the fact that these pathogens are a serious threat to forestry worldwide, both in natural ecosystems and plantations (Davidson et al. 2003; Garbelotto et al. 2003; Greslebin and Hansen 2010). The appearance of a needle blight of Pinus spp. caused by a Phytophthora sp., as has occurred with P. pinifolia was, however, unprecedented and unexpected.
The first pathogenicity tests conducted with P. pinifolia using mycelial plugs showed clearly that P. pinifolia was able to infect and cause disease in the shoots in a period of less than 30 days (Durán et al. 2008). However, the typical DFP symptoms observed in the field have never been artificially reproduced. In this study, we confirm the results from previous mycelial inoculation studies regarding the pathogenicity of P. pinifolia, and extend these to additional Pinus spp. that are of importance as potential plantation species in Chile and other parts of the world. The primary aim was, however, to produce sporangia and zoospores of P. pinifolia and to attempt to reproduce the characteristic resinous bands on the foliage of P. radiata, to re-isolate the organism from the infected needles, completing Koch’s rules of proof for the organism. The focus on infection propagules also provided a parallel opportunity to study the means of dispersal of P. pinifolia, by monitoring sporangial production and the rainfall across different areas.
Materials and methods
Isolates and inoculum production
The three isolates used throughout the study were selected from a previous investigation where six isolates were inoculated into 180 1-year-old P. radiata plants (30 per isolate) and where it was shown that they were all pathogenic and did not differ in their relative levels of aggressiveness (R. Ahumada, unpublished). One isolate (CMW33986) of P. pinifolia was used in the first experiment with mycelial plugs and three isolates (CMW33983, CMW33986 and CMW34012) in the second experiment using suspensions of sporangia and zoospores. All the isolates were obtained from needles of P. radiata with the resinous bands characteristic of infection by P. pinifolia. The three isolates selected for the inoculations were chosen because they were similar in relative aggressiveness in a previous study (R. Ahumada, unpublished).
Isolates were identified based on culture morphology on the selective medium CARP (Hansen and Hamm 1996) and using the species-specific primers developed by Durán et al. (2009). Emerging colonies were transferred to Carrot Agar (CA) (Erwin and Ribeiro 1996) and maintained between 18 and 22 °C for 20 days until they could be identified. All three isolates have been maintained in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa.
For inoculum production, 20 replicates of each isolate were grown in V8 agar at 22 °C for 2 weeks. From the edges of actively growing cultures, 5 discs (7 mm diam.) were transferred to a 60 mm Petri dish containing 25 ml of 10 % V8 broth and incubated for 24 h at 22 °C (Erwin and Ribeiro 1996). The agar discs were washed twice with autoclaved cold distilled water, and then immersed in filtered pond water for 48 h at 22 °C under continuous cool white fluorescent light (6,600 lx). Isolates were examined for the presence of sporangia and those bearing these structures were chilled at 4 °C for 2 h to induce the release of zoospores. The primary zoospore suspension was poured into a sterile beaker and maintained at 4 °C until inoculation. Three aliquots of 10 μL were taken from the beaker and used to determine the zoospore concentration using a hematocytometer. The final zoospore suspension was prepared by adding autoclaved distilled water and adjusting this to approximately 5 × 104 zoospores ml−1. The zoospore suspension was maintained at 4 °C and transported to the field for inoculation on the same day. To determine the viability of the zoospores, three aliquots of 30 μL of the suspension were sampled in the field after the inoculation and spread onto CARP, transported to the laboratory at 4 °C, incubated at 22 °C for 2 weeks and evaluated for growth.
The inoculations were performed in two different experiments, under laboratory conditions and in the field. The laboratory trials were conducted at Bioforest research facilities in Concepción. The screening facility was set to provide a photoperiod of 12 h of artificial light, at 75 % relative humidity, a temperature of between 18 and 22 °C and the containers were irrigated daily for 1 h.
The field experiment was installed on the Llico farm (37°22′45″S; 73°58′W; Arauco province). A shade house (70 % shade netting) was erected for this inoculation study and a fogging system was installed and applied for 15 min, three times per day (10, 14 and 18 h) to maintain a high level of humidity over the foliage of the inoculated trees. The shade house was surrounded by a plantation of Eucalyptus globulus trees so as to reduce any chance of natural infection by P. pinifolia.
Inoculation of Pinus spp. with P. pinifolia mycelium plugs
Twenty plants (15-month-old, an average of 22 cm tall and 0.8 cm average diam. at the substrate level) of each of 13 Pinus spp. including varieties [P. arizonica (PAR), P. durangensis (PDU), P. greggii mix of families (PGR1), P. greggii var australis (PGR2), P. greggii var greggii (PGR3), P. maximinoi (PMA), P. muricata (PMU), P. patula mix of families (PPA1), P. patula var longipedunculata (PPA2), P. patula var patula (PPA3), P. pinaster (PPI), P. radiata (PRA) and P. taeda (PTA)] were established in 140 cc containers. The plants were acclimatized for 2 weeks prior to inoculation in the screening facilities using the conditions described above. Small bark discs (4 mm diam.) were removed from succulent tissue of the stems, between 10 and 12 cm from the growing tips and a plug of mycelium (3-week-old culture of P. pinifolia grown on CA) of similar size was placed into the wounds. Discs of clean CA were used as negative controls. Fifteen plants of each species and the varieties were inoculated with the isolate CMW 33986 of P. pinifolia and five plants per species were inoculated as controls. Inoculation wounds were covered with Parafilm to reduce desiccation and contamination. Four weeks after inoculation, the Parafilm and the bark around the inoculation points was removed with a sterile scalpel and the lesion lengths were recorded.
Re-isolation was attempted from the leading edges (top and bottom) of the lesions on all inoculated plants. Small pieces (<5 mm2) of succulent infected tissue from the leading edges of the lesions were plated onto CARP medium to re-isolate the inoculated organism and ensure that it was associated with the lesions. The identity of 10 % of the resulting cultures that were characteristic of P. pinifolia was confirmed using the PCR specific primers (Durán et al. 2009).
Analysis of data was conducted separately for all Pinus species, using the linear model of analysis of variance (ANOVA) and means were separated based on LSD (Least Significant Difference) using the software Statistica V9 for Windows (Stat Soft Inc 2004).
Inoculation of P. radiata with P. pinifolia zoospores
This experiment was conducted with a suspension of zoospores and sporangia using a mixture of the three isolates as described above. An equivalent volume of zoospore suspension (adjusted to approximately 5 × 104 zoospores ml−1) was transferred to forty 2 L plastic bags, in which individual P. radiata plants were immersed (75 % of their foliage) overnight (~12 h). After immersion, half of the plants (20) were placed in the screening facility in the laboratory and the other half (20) transported in a cooler box, to the shade house in the field on the Llico farm. Forty plants, with the same characteristics as those inoculated, were used as controls and were dipped into sterile distilled water overnight. Half of the plants were placed in the laboratory in an area separate from the plants inoculated with P. pinifolia, to avoid cross contamination and the other half were transported to the field and placed in the shade house together with the inoculated plants. All plants were inspected weekly for the appearance of symptoms.
Four weeks after the inoculation, symptoms were recorded on all inoculated plants. Evaluations included the presence (1) or absence (0) of resinous bands on the needles of both inoculated and control plants. A needle was considered to be infected if it had at least one resinous band. A sample (~10 %) of the bands produced on the needles was used for re-isolation of the inoculated pathogen using the same methodology described above for plants inoculated with mycelial plugs.
A Bayesian approach was used for data analyses. The variable response was assumed to be a binomial random variable and the percent of infection was set to a beta prior distribution. The analyses were separated between the plants in the laboratory and the plants maintained in the field, in order to avoid possible confounding effects in the responses measured. Bayesian credible intervals for each proportion (π) were computed using Win BUGS (Bayesian Inference Using Gibbs Sampling) software (Lunn et al. 2000). The Gibbs sampling chain was run for 11,000 iterations with the first 1,000 iterations used as burn-in. The convergence of the Gibbs chains was checked using the Heidelberger and Welch’s test (Heidelberger and Welch 1983).
Fluorescence microscopy was used to provide an additional confirmation of the presence of P. pinifolia on the foliage. A sub-sample of 10 % of the needles with resinous bands were immersed in a solution of Calcofluor Fluorescent Brightener 28 (Sigma-Aldrich, St. Louis) at 0.001 % in 0.05 M Tris-HCl (pH 8.0) for 30 s. These needles were mounted on glass slides and examined under a fluorescence microscope (Olympus CX31, Olympus America Inc.) for the presence of sporangia or mycelium on the needle surfaces.
Monitoring the presence of sporangia in plantations
Six field sites were selected for monitoring the presence of P. pinifolia inoculum in a natural environment. This monitoring was carried out between May 2009 and December 2011. The selected sites were located in three 7-year-old P. radiata plantations that have been historically infected with P. pinifolia. Each monitoring site consisted of a block of approximately 100 m2 (20 × 5 m) from which 10 trees with typical symptoms of P. pinifolia damage were selected. From each tree, 20 needles were sampled twice each month and transported to the laboratory for analysis. In the laboratory, a minimum of 50 needles from the total collection (~200 per site) having clear resinous bands were selected for detailed examination. For each needle, a section of approximately 4 cm was cut around the resinous band. These needle pieces were immersed in a solution of Calcofluor Fluorescent Brightener 28 for 30 s and then mounted on glass slides for microscopic observations. All observations were made using an Olympus fluorescence microscope as above, to quantify the presence of inoculum (sporangia, zoospores and mycelium) on the needle surface. The sections of every needle examined corresponded to approximately 30 stomata and thus, approximately 18,000 stomata (30 stomata × 50 needles × 6 areas × 2 sampling periods) were examined each month.
The data recorded using fluorescence microscopy were tabulated and then plotted as monthly average of sporangia per needle (or per resinous band abbreviated as RB). The monthly rainfall was also recorded at the sampling sites during the same period of observation. The average of inoculum (sporangia) per month was plotted together with the rainfall for the entire evaluation period.
Inoculation of Pinus spp. with P. pinifolia mycelium plugs
Phytophthora pinifolia was consistently re-isolated from the lesions and the identity of the cultures selected was confirmed in 100 % of the cases using PCR-specific primers. None of the control plants in the all species treated, showed any evidence of symptoms and P. pinifolia could not be isolated from them.
Inoculation of P. radiata with P. pinifolia zoospores
Incidence of P. pinifolia symptoms on P. radiata plants inoculated with zoospores and maintained in the laboratory or under field conditions
More than 95 % of the isolations made from the resinous bands yielded isolates having characteristic P. pinifolia morphology and those analysed with PCR specific-primers were all confirmed to be of this organism. It was possible to consistently produce the necessary amount of inoculum for the all experiments and zoospores were shown to have an average of 86 % viability.
Monitoring the presence of sporangia in plantations
While it has been relatively certain that P. pinifolia causes the needle disease DFP on P. radiata in Chile, it has never been shown experimentally that this organism can cause symptoms identical to those associated with natural infections. Results of this study have resolved this problem and have completed Koch’s postulates, showing unequivocally that P. pinifolia causes DFP on P. radiata.
In this study, where P. radiata tissue was immersed in zoospore/sporangial suspensions, infection occurred only on needles and never on the succulent stem tissue. This is an important observation because green stem tissue is commonly seen to be infected under field conditions (Durán et al. 2008). Results of this study also confirm that natural infection occurs mainly via the needles and that P. pinifolia moves down the needles to infect the stem tissue. Despite these observations, it is not clear whether the succulent tissue of young stems can be infected directly although this seems not to be the case from the results of the present study.
Pathogenicity tests made by inoculating stems of 13 different Pinus spp. and varieties with mycelial plugs, showed a significant variation in the susceptibility of the species tested, from apparently resistant species such as P. pinaster and P. taeda to highly susceptible species including P. durangensis, P. radiata and P. arizonica. Despite the fact that P. pinifolia caused infections on various Pinus spp., operational field monitoring carried out since 2006 has never found evidence of infection in other plants such as Nothofagus spp., Eucryphia cordifolia, Luma apiculata, Chusquea quila, Aristotelia chilensis, which are very common in the area (unpublished data). This is in contrast to many other Phytophthora spp., such as P. ramorum that has a wide range of hosts (Davidson et al. 2003; Rizzo et al. 2005; Tooley and Browning 2009). The fact that P. pinifolia has been found only in Chile and its low genetic diversity (Durán et al. 2010) suggests strongly that it is a pathogen specific to Pinus spp.
Stem inoculation with mycelial plugs gave similar results to those observed in a previous study (Durán et al. 2008). The inoculated seedlings showed a general chlorosis of the needles, but did not produce the characteristic symptoms on the needles. The stem inoculations were, however, substantially more efficient in terms of the time needed to prepare inoculum and the evaluation of symptoms. Lesion lengths were easily measured and showed a clear tendency in terms of susceptibility. This inoculation method has been used many times as a test of the susceptibility of different hosts to Phytophthora spp. (Rizzo et al. 2002; Hüberli et al. 2002; Oh et al. 2006; Durán et al. 2008; Greslebin and Hansen 2010) and appears to offer a reliable means to estimate broad differences in susceptibility of Pinus spp.
Phytophthora pinifolia is unlike other aerial Phytophthora spp. where sporangia are caducous and serve as the major dispersal propagules. This is for example the case of P. ramorum, P. kernoviae, P. cactorum, P. infestans and P. capsici, which typically produce caducous sporangia, as the main propagules for dissemination (Erwin and Ribeiro 1996; Hansen et al. 2003; Brasier et al. 2005; Rizzo et al. 2005). Results also showed that sporangia are produced prolifically from the stomata of infected needles and specifically from the areas where the typical resinous bands are present. Empty sporangia can be seen suggesting that zoospores are released from the intact sporangia on the needles. The sporangiophores appear to twist and break at some distance from the sporangia, although this was observed only occasionally. The strong coastal winds and the rain-splash during the rainy season on the coast of the Biobío region of Chile would facilitate this form of dispersal. This suggests that these structures can also represent a means of spread for P. pinifolia but our observations suggest that this happens infrequently and it is a question that deserves further study.
The zoospore/sporangia inoculation technique used in this study was technically challenging and is unlikely to be suitable in its current form for large scale experimentation and resistance screening. This is in contrast with other systems, such as those of Hansen et al. (2005) who found that leaf-dip inoculation represented a rapid and reliable method to predict susceptibility to P. ramorum infection. In the present study, inoculation with zoospores reproduced the typical symptoms of Daño Foliar del Pino, but it was variable and inconsistent in the level of infection observed. For example, the number of resinous bands produced per plant in the laboratory was different to that seen under field conditions, although these differences were not statistically significant. In contrast with P. ramorum, which is very easy to inoculate by spraying of zoospores (P. Resser, pers. comm.), this has not been achieved with P. pinifolia thus far. It will thus be important to develop a more complete understanding of the factors influencing infection by P. pinifolia, in order to standardize the protocols for resistance screening.
Detailed monitoring of sporangial development highlighted key aspects of the biology and epidemiology of P. pinifolia. It was evident that sporangial development was closely linked to rainfall patterns. Not only did sporangial development correlate with the onset and seasonal variation of rainfall, but it was also remarkably closely linked to the amount of rainfall in a particular period of time. The same patterns have also been observed with P. ramorum in California and Oregon, which has a Mediterranean climate with a distinct wet and dry season, and sporulation of the pathogen with subsequent plant infection appear to be primarily restricted to the rainy season (Davidson et al. 2005; Rizzo et al. 2005).
This study allowed a deeper understanding of P. pinifolia as the cause of the Daño Foliar del Pino on P. radiata in Chile, because it was possible to reproduce symptoms identical to those observed in natural infections. The pathogenicity tests showed that P. pinifolia is not specific to P. radiata and was able to infect many of the other Pinus spp. and hybrids that were artificially inoculated. It was also shown that two of the 13 tested species (P. pinaster and P. taeda) appeared to be most resistant. In the case of P. pinaster, this confirmed field observations where the species shows no symptoms when grown close to heavily infected P. radiata trees. The sporangial monitoring demonstrated that propagules of P. pinifolia remain on the foliage throughout the year, but their presence is very closely correlated with climatic conditions, of which rainfall is most important.
We thank Bioforest S.A. and Bosques Arauco, subsidiaries of Arauco Group for their financial and technical support. We also thank various colleagues including Mr Rolando Gómez, Mr Rafael Quezada, Mr Rodrigo Nova, Dr Alvaro Durán, Mrs Carolina Valiente and Mr Boris Palma for their commitment in the field and laboratory work together with the Lab workers. Mrs Marcela Millar and Mr Iván Appel to provide the Pinus species tested. Mr Eduardo Rodríguez and Dr Claudio Balocchi, for useful discussions that have assisted us in completing this study. We also thank Mrs Seonju Marincowitz from University of Pretoria and Mr Freddy Mora from Universidad de Concepción for their support and help with microscope measurements and statistical analyses, respectively.
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