Detection of the Diplodia shoot blight and canker pathogens from red and jack pine seeds using cultural methods

Abstract

The shoot blight and canker pathogens Diplodia pinea and D. scrobiculata commonly and abundantly sporulate on seed cones of red pine (Pinus resinosa) and jack pine (P. banksiana) collected from Wisconsin and Minnesota forests. Cultural methods were used to investigate the incidence of these fungi in seed lots obtained from government nurseries in these states. In each of three replicate trials, seeds of each lot were assigned to four treatments before incubation on semi-selective medium: (1) not surface-disinfested; (2) surface-disinfested; (3) surface-disinfested, then inoculated with D. pinea conidia; or (4) not surface-disinfested, then inoculated with D. pinea conidia. For red pine seeds, the mean percentage positive was 2.7% for treatment 1 and 1.3% for treatment 2. Jack pine seeds were less frequently positive than red pine seeds for both treatments 1 and 2. The Diplodia species cultured was identified as D. pinea in almost every case. Diplodia pinea was much less frequently recovered from seeds that were not surface-disinfested and then inoculated (treatment 4), when compared with seeds that were inoculated with D. pinea after surface-disinfestation (treatment 3). Results confirm the potential for dissemination of D. pinea on red pine and jack pine seeds, and caution is warranted before concluding absence of the pathogen based on results using cultural methods with relatively small numbers of seeds. Although the frequency of pathogen-positive seeds was low, the large numbers of seeds planted in nurseries suggest that seeds may be a potentially important route of entry of D. pinea into nursery beds.

Résumé

Les agents pathogènes Diplodia pinea et D. scrobiculata, causant la brûlure des pousses et le chancre, sporulent couramment et abondamment sur les cônes de pin rouge (Pinus resinosa) et de pin gris (P. banksiana) collectés dans les forêts du Wisconsin et du Minnesota. Des méthodes culturales ont été utilisées pour évaluer l’incidence de ces champignons dans les lots de semences obtenus de pépinières gouvernementales de ces États. Dans chacun de trois essais répétés, les semences de chaque lot ont été soumises à quatre traitements avant incubation sur milieu semi-sélectif: (1) leur surface n’a pas été décontaminée; (2) leur surface a été décontaminée; (3) leur surface a été décontaminée puis inoculée avec des conidies de D. pinea; ou (4) leur surface n’a pas été décontaminée, mais elle a été inoculée avec des conidies de D. pinea. En ce qui a trait aux semences de pin rouge, le taux d’infection résultant du traitement 1 était de 2.7 % et de 1.3% à la suite du traitement 2. Les semences de pin gris étaient moins souvent infectées à la suite des traitements 1 et 2 que celles de pin rouge. Dans la majorité des cas, l’espèce de Diplodia cultivée a été identifiée comme D. pinea. Ce dernier était moins souvent collecté sur les semences dont la surface n’avait pas été décontaminée mais inoculée (traitement 4), comparativement aux semences qui avaient été inoculées avec D. pinea après que leur surface avait été décontaminée (traitement 3). Les résultats confirment le potentiel de dissémination de D. pinea sur les semences de pin rouge et de pin gris et qu’il faut faire preuve de prudence avant de conclure à l’absence de l’agent pathogène en fonction de résultats obtenus par méthodes culturales lorsque des nombres relativement faibles de semences sont utilisés. Bien que la fréquence des semences infectées par des agents pathogènes fût faible, le grand nombre de semences plantées dans les pépinières suggère que ces semences peuvent être une voie d’introduction possiblement importante de D. pinea dans les planches des pépinières.

Keywords:

• Diplodia
• pine
• Pinus banksiana
• Pinus resinosa
• seed infestation
• Sphaeropsis

Mots clés:

• contamination des semences
• Diplodia
• pin
• Pinus resinosa
• Pinus banksiana
• contamination des semences
• Sphaeropsis

Introduction

Diplodia pinea (Desm.) J. Kickx fil. and the more recently described species D. scrobiculata J. de Wet, B. Slippers & M. J. Wingf. cause shoot blight, cankers, and collar rot of red pine (Pinus resinosa Aiton) and jack pine (P. banksiana Lamb.) seedlings. Before recognition of D. scrobiculata as a distinct species (De Wet et al. 2003), these fungi were referred to as morphological types of Diplodia pinea. The known geographic and host ranges of D. pinea are much greater than those of D. scrobiculata, and severe damage in much of the world where pines occur naturally or have been introduced usually is attributable to D. pinea (sensu stricto). Therefore, unless specifically identified as D. scrobiculata in this paper or previous reports cited herein, the name D. pinea will be used in this paper for various synonyms of D. pinea, including Sphaeropsis sapinea and S. ellisii.

High incidence and severity of Diplodia shoot blight in bare-root seedling nurseries in Minnesota and Wisconsin historically has been associated with inoculum disseminated from infested red pine windbreaks adjacent to seedling beds (Palmer et al. 1988; Stanosz et al. 2005). Diplodia pinea can also persist on or in asymptomatic seedlings and later proliferate to cause disease symptoms, including mortality, under conditions that induce host stress (Stanosz et al. 1997, 2001). In response to losses in nurseries and after outplanting, disease management practices including red pine windbreak removal have been implemented in state government-operated bare-root nurseries in Minnesota and Wisconsin. Although disease incidence is much lower than previously reported, D. pinea continues to be present in these nurseries (Minnesota Department of Natural Resources 2009; Wisconsin Department of Natural Resources 2012).

Diplodia pinea has been associated with seeds of many pine species. Birch (1936) reported that seeds of Austrian pine (P. nigra Arnold), Monterey pine (P. radiata D. Don), and ponderosa pine (P. ponderosa Lawson and C. Lawson) carried D. pinea. Fungi identified as D. pinea (or synonyms) or only to the genus Diplodia were cultured from seeds of Bhutan pine (P. wallichiana A. B. Jacks.) (Ashaeer-Farooq 2000), Caribbean pine (P. caribaea Morelet), michoacan pine (P. devoniana Lindl.), ocote chino (P. oocarpa Schiede ex Schltdl.) (Rees & Webber 1988), loblolly pine (P. taeda L.) (Huang & Kuhlman 1990), Mexican weeping pine (P. patula Schiede ex Schltdl. & Cham.) (Smith et al. 1996, 2002), Monterey pine (P. radiata) (Smith et al. 1996; Bihon et al. 2011a) and slash pine (P. elliottii Englem.) (Anderson et al. 1984; Huang & Kuhlman 1990; Fraedrich et al. 1994; Fraedrich & Miller 1995).

Diplodia pinea or D. scrobiculata frequently colonize red and jack pine cones. Either fungus was present on cones collected from the forest floor at 106 of 109 red pine stands and all of 28 jack pine stands in Minnesota (Albers et al. 2008). In neighbouring Wisconsin, at least one of these species was present on ≥84% of red pine cones and ≥42% of jack pine cones collected from canopies of trees in mixed pine stands (Munck & Stanosz 2009). Also in Wisconsin, D. pinea was reported to be present on 84% of cones from a red pine windbreak (Nicholls & Ostry 1990), and on all red pine cones collected from debris following plantation harvests (Oblinger et al. 2011). Using water-extraction techniques, large numbers of viable conidia can be extracted from pycnidia borne on these cones (Munck & Stanosz 2009; Oblinger et al. 2011). Despite association with cones, the occurrence of these fungi on or in seeds of red and jack pines has not yet been determined.

Red and jack pine seeds to be planted in forest tree nurseries are extracted from cones collected in forests or produced in seed orchards. The common occurrence and abundant sporulation of D. pinea and D. scrobiculata on red and jack pine cones suggest that seed might carry these fungi, and thereby serve as a source of inoculum responsible for continued presence in seedling beds (Minnesota Department of Natural Resources 2009; Wisconsin Department of Natural Resources 2012). The objective of this study was to determine the frequency of internal and external contamination of red and jack pine seeds by these pathogens. Laboratory methods similar to those developed for culture of D. pinea and D. scrobiculata from asymptomatic seedlings were used (Blodgett et al. 2003). Molecular methods were used to confirm pathogen identity.

Materials and methods

Source of seeds

Seeds were obtained from state government nurseries in Minnesota (three red pine seed lots) and Wisconsin (five red pine seed lots and five jack pine seed lots). At the Minnesota Department of Natural Resources (DNR) Badoura Nursery, red pine seed is extracted after heating the cones at 66–71 °C for 1.5 h. Seed is then dewinged, cleaned and stored at <10% moisture content at 2 °C in 26.5 L metal cans with screw-top lids sealed with paraffin wax. At the Wisconsin DNR Hayward Nursery, cones of both red and jack pines are dried at approximately 59 °C for 9 h. Extracted seed is dewinged, cleaned and then stored at ≤6% moisture content at −7 °C in plastic bags placed within foil-lined paper barrels.

Treatments and cultural methods

In each of three separate trials, seeds of each lot were assigned to four treatments: (1) not surface-disinfested, 100 seeds; (2) surface-disinfested, 50 seeds; (3) surface-disinfested and then inoculated with D. pinea conidia, 50 seeds; or (4) not surface-disinfested, but inoculated with D. pinea conidia, 50 seeds. Seeds to be surface-disinfested (treatments 2 and 3) were placed in a perforated plastic vial (each lot and treatment combination in a separate vial), rinsed briefly under cold tap water, stirred for 10 min in cold tap water, and then stirred for 4 h in 10% hydrogen peroxide, followed by three rinses of 10 min each in sterile deionized water. The seeds were then dried on sterile aluminium foil in a laminar flow hood, with each lot and treatment on a separate piece of foil.

Pycnidia of D. pinea isolate 411 that had been obtained from red pine were produced on autoclaved red pine needles placed on colonies of the fungus growing on water agar. Conidia were extracted in sterile water and stored in sterile water at −80 °C until used. Seeds were inoculated (treatments 3 and 4) by placing a 3 μL drop of conidial suspension (2.8 × 10⁴ spores mL⁻¹ for MN seed and 5.1 × 10⁴ spores mL⁻¹ for Wisconsin seed) on each seed, which was then allowed to dry in the laminar flow hood. Germination of conidia on water agar was ≥68% for each trial.

Seeds were placed into 20-mm-diameter by 150-mm-long culture tube slants containing approximately 10 mL of tannic acid (TA) medium (0.5% tannic acid and 2% agar) (Blodgett et al. 2003) and autoclaved red pine needles. After 6 weeks incubation at room temperature under white and black fluorescent lights, the needles were checked for presence of pycnidia with conidia consistent with those of Diplodia species (Punithalingam & Waterston 1970). For each lot and treatment combination, the mean for the three trials of the percentage of seeds from which a Diplodia colony was detected was calculated. In addition, for each seed lot separately, paired t-tests were used to compare the frequencies of detection for treatments 3 and 4. Percentage data were converted to proportions and the arcsine of the square root transformation was applied before performing this analysis using Minitab for Windows version 14 (Minitab Inc., State College, PA).

Confirmation of pathogen identity

Molecular methods were used to confirm the identity of the Diplodia species that were detected in treatments 1 and 2. Transfers were made to potato dextrose broth and after 7 days incubation DNA was extracted and species-specific PCR primers were used as described by Smith and Stanosz (2006). It was outside the scope of this study to identify or enumerate fungi other than D. pinea and D. scrobiculata, and the TA medium is somewhat selective for these Diplodia species (Blodgett et al. 2003). Therefore, no attempt was made except to record presence of some common genera of fungi that sporulated in the culture tubes; identification was based on morphology.

Results and discussion

Although D. pinea and D. scrobiculata have been known to commonly occur on red and jack pine cones (Palmer et al. 1988; Munck & Stanosz 2009), to our knowledge this is the first study during which these pathogens were detected from seeds of these trees. Vujanovic et al. (2000) studied fungi of cones and seeds collected from 18 conifer species growing in a botanic garden. Isolation of D. pinea from 12 species (including red pine) was reported, but whether the source of isolates was red pine cones or seeds was not indicated. Knowledge of the association of D. pinea and D. scrobiculata with red and jack pine seeds contributes to a more complete understanding of the relationships between these fungi and these host trees, with implications for interpretation of past research and future efforts to minimize disease development and further pathogen spread.

The presence of D. pinea or D. scrobiculata was recorded from non-inoculated seed in seven of the eight red pine seed lots and three of the five jack pine seed lots. Frequencies of positive seeds were very low for seeds that were not surface-disinfested (treatment 1), and also for seeds that were surface-disinfested (treatment 2) (Table 1); therefore, statistical comparisons among results obtained for these treatments were not attempted. For all red pine lots, mean percentages of positive seeds that had not been surface-disinfested were <5%, and were <3% for surface-disinfested seeds. For both of these treatments, mean percentages of positive jack pine seeds were <1.3%, and often were 0%.

Table 1. Percentages of red pine and jack pine seeds from Minnesota (MN) and Wisconsin (WI) nurseries from which Diplodia shoot blight pathogens were detected on agar medium.

TREATMENT^a
Host, state and seed lot number	None^b	Surface disinfested^c	Not surface disinfested D. pinea added^c	Surface disinfested D. pinea added^c	P-value
Red pine
MN 1	1.3	0.6	92.7	54.0	0.03
MN 2	3.7	0.6	90.0	49.3	0.04
MN 3	0.0	0.0	92.7	76.0	0.07
WI 1	3.3	2.7	96.0	19.3	<0.01
WI 2	1.7	1.3	92.0	34.6	<0.01
WI 3	2.3	0.0	92.0	36.0	0.02
WI 4	4.7	2.0	94.0	31.3	<0.01
WI 5	4.7	2.0	88.7	34.0	0.11
Jack pine
WI 6	0.3	0.0	89.3	19.3	<0.01
WI 7	0.7	0.0	90.0	54.0	0.04
WI 8	0.0	1.3	84.7	12.7	0.03
WI 9	0.0	0.0	96.0	24.0	<0.01
WI 10	0.0	0.0	96.7	14.7	0.02

^aFor each seedlot separately, results were compared for seeds either surface-disinfested or not surface-disinfested, to which D. pinea conidia were then added. Paired t-tests were used after conversion of percentages to proportions and arcsine of the square-root-transformation.

^bValues are mean percentages for three separate trials of 100 seeds each, incubated without treatment.

^cValues are mean percentages for three separate trials of 50 seeds each, incubated with or without surface-disinfestation and with or without subsequent addition of D. pinea conidia.

The previously reported frequencies of association of D. pinea (or its synonyms) with seeds of pine species have varied widely, and appear to be influenced by the methods employed. At one extreme, Birch (1936) reported from New Zealand that D. pinea was present on 90–100% of P. radiata seed extracted from ‘Diplodia-infected cones’. In South Africa, Smith et al. (2002) reported isolation of D. pinea from 23% of Mexican weeping pine seeds and seed wings that had not been surface-disinfested. As we found in the current study, however, percentages of pine seeds from which D. pinea or unidentified Diplodia species were detected are generally much lower. Birch (1936) also reported that D. pinea was isolated from 4% of surface-disinfested seeds of P. nigra var. calabrica (syn. P. nigra J. F. Arnold subsp. laricio Maire). Although he found that while D. pinea was present in all samples of several consignments of imported ponderosa pine seeds, the frequency of ‘infected seed’ determined after surface disinfestation never exceeded 0.2%. In South Africa, Bihon et al. (2011a) isolated D. pinea from 2.3% of Monterey pine seeds, and 0% of Mexican weeping pine seeds that were first surface-disinfested. Fraedrich et al. (1994) studied factors affecting frequency of fungi cultured from seeds of slash pines in a seed orchard in Florida. They reported that D. pinea was among the fungi that were less frequently cultured from seeds obtained from cones collected off trees and cones dried promptly after collection, than from seeds obtained from cones on the ground and from cones stored for 5 weeks before drying.

For inoculated seeds, mean percentages from which D. pinea or D. scrobiculata were detected differed greatly depending on whether seeds were first surface-disinfested (treatment 3) or were not (treatment 4) (Table 1). For all lots of both pine species, means for percentages of positive seeds that had been surface-disinfested and then inoculated were >84%. Mean percentages of positive seeds that were inoculated, but were not first surface-disinfested, were as low as 19.3% for one red pine seed lot and as low as 12.7% for one jack pine seed lot. Results from these two treatments for each seed lot of each host species separately were significant (P ≤ 0.04) for all but two seed lots. This finding suggests that on non-surface disinfested seed, the occurrence of existing fungi reduced or competed with the D. pinea that was inoculated.

The relatively infrequent detection from surface-disinfested seeds suggests that Diplodia shoot blight fungi rarely penetrate red and jack pine seed coats, at least for seeds that have been placed into storage after routine procedures employed for extraction, dewinging and cleaning. However, fungi identified as belonging to the genera Aspergillus, Penicillium, Rhizopus and Trichoderma that commonly sporulated in the culture tubes containing seeds which were not surface-disinfested (treatments 1 and 4) could have masked the presence of D. pinea. Blodgett et al. (2003) found that radial growth rates of D. pinea and D. scrobiculata on agar medium amended with tannic acid were less inhibited compared with those of several other fungi. But numerous taxa still grew on this medium when re-isolation of D. pinea from inoculated red pine branches was attempted. The efficiency of recovery of Diplodia pathogens on this medium is unknown. Alternatively, the differences in results obtained for treatments 3 and 4 may be attributable to some physical or chemical effect of the surface-disinfestation treatment that favoured proliferation of D. pinea on the treatment 3 seeds. Future studies could utilize both isolation methods and molecular techniques that might facilitate detection of these pathogens from seed, especially when rare or in the presence of other fungi. A PCR-based method has been successfully used to detect D. pinea and D. scrobiculata directly from bark and wood of red and jack pine seedlings exhibiting collar rot symptoms at field sites (Smith & Stanosz 2006), and might be useful for this purpose. Amplification of DNA from dead mycelium or spores, however, could lead to erroneous conclusions regarding presence or abundance of a viable pathogen and the risk of introduction.

Seeds of red pine and jack pine differed in the frequency of detection of the two Diplodia species from seeds that had not been inoculated (treatments 1 and 2). Using species-specific PCR primers to identify cultures obtained from these red pine seeds, D. scrobiculata was detected only once. Diplodia pinea was the pathogen obtained from all of the remaining 79 culturally positive, non-inoculated red pine seeds. In contrast, D. scrobiculata was identified from five of the six culturally positive jack pine seeds. The observed differences in the frequencies of detection of D. pinea and D. scrobiculata from seeds of the red and jack pines are consistent with other reported differences in the relationships of these fungi with these two hosts. Munck and Stanosz (2009) reported that these fungi were present more often on red pine cones than on jack pine cones collected from tree canopies or the ground under trees in the mixed pine stands in Wisconsin. The proportion of cones for which molecular methods identified D. pinea or D. scrobiculata also differed by host, with a tendency for the former to be detected more frequently from red pine cones, and the latter more frequently from jack pine cones. Similarly, in a study of dead, naturally occurring seedlings, D. pinea was more frequently identified from red pine seedlings than D. scrobiculata, which was more frequently identified from jack pine seedlings than D. pinea (Smith & Stanosz 2006). And finally, both incidence and severity of disease resulting from inoculation with conidia of D. pinea were greater on red pine seedlings than on jack pine seedlings (Blodgett & Stanosz 1997). The basis for these apparent host preferences of the pathogens and exploitation of possible differences in resistance of these two hosts to reduce damage due to Diplodia shoot blight have not yet been explored.

Direct effects of Diplodia shoot blight fungi on seed vitality and germination have been documented elsewhere. Although results varied among isolates, Rees and Webber (1988) reported 49% germination of false Weymouth pine (P. pseudostrobus Lindl.) seeds inoculated with an isolate and stored for 2 months at 2 °C, compared with 69% germination of non-inoculated, stored seeds. They also reported apparent isolate and host effects on mortality of 14- to 20-day-old seedlings of three Central American pine species placed on cultures of D. pinea, with death of up to 98% of false Weymouth pine seedlings. Effects of D. pinea inoculation and storage on germination of Monterey pine seeds were also reported (Bihon et al. 2011a). Only 53% of seeds germinated after inoculation with a conidial suspension and storage for 20 days at room temperature before transfer to plates of water agar, compared with 76% of inoculated seeds plated the day after inoculation and 91% germination of non-inoculated seeds. Inoculation of red pine and ponderosa pine seeds with D. pinea reduced germination by 11% (compared with non-inoculated seeds), and also resulted in 11% and 27% radicle decay, respectively (Fisher 1941).

Disease also can develop following transfer of seed-borne shoot pathogens to young pine seedlings. The pitch canker pathogen Gibberella circinata (syn. Fusarium subglutinans f. sp. pini) has been isolated from diseased Monterey pine seedlings grown from naturally infested seeds (Storer et al. 1998) and artificially infested seeds (Aegerter & Gordon 2006). Sutherland et al. (1981) isolated the shoot blight fungus Sirococcus conigenus (syn. S. strobilinus) from dead spruce seedlings (Picea spp.) grown from naturally infested seed. The authors attributed initial Sirococcus shoot blight outbreaks in coastal British Columbia container nurseries to seed-borne inoculum, and mortality from secondary spread was reported to reach 30% in some seedlots (Sutherland et al. 1989). Similar potential has not yet been proven for D. pinea or D. scrobiculata. Bihon et al. (2011a) did not isolate D. pinea from Monterey pine seedlings grown from seeds inoculated with a conidial suspension, but only 10 seeds were used in that study.

Clearly, great caution is warranted before concluding that Diplodia shoot blight pathogens are absent from seed samples, especially when that conclusion is based on results using cultural methods with relatively small numbers of seeds bearing their natural microflora. Seed transmission is the likely explanation for the long history, wide geographic distribution and common occurrence of D. pinea wherever pines are cultivated, and the more recent discovery of D. scrobiculata in South Africa (Bihon et al. 2011b). Repeated, worldwide movement of large numbers of seeds of many coniferous species for nursery seedling production may also explain the relative similarity of some isolates among collections of D. pinea from diverse hosts on multiple continents (Stanosz et al. 1999), and diversity among isolates where this pathogen has been introduced (Burgess & Wingfield 2002). Recent reports of the expansion in the known distribution of D. pinea to include Estonia (Hanso & Drenkhan 2009) and Sweden (Oliva et al. 2013) reflect the continuing threat of pathogen introduction, and need for vigilance and restriction on movement of infested seed and other plant materials.

Acknowledgements

The provision of seed by Craig Van Sickle and Gordon Christians, supervisors of the Minnesota Department of Natural Resources nursery in Badoura, MN and Wisconsin Department of Natural Resources nursery in Hayward, WI, respectively, is gratefully acknowledged. These agencies also provided partial funding to support this study.