Surveys for asymptomatic persistence of Sphaeropsis sapinea on or in stems of red pine seedlings from seven Great Lakes region nurseries
Etude de la persistance asymptomatique de Sphaeropsis sapinea sur ou dans les pousses de semis de Pin rouge (Pinus resinosa) dans sept pépinières de la région des Grands Lacs
Symptomlose, epi- und endophytische Besiedlung von Stämmen von Sämlingen der Amerikanischen Rotkiefer (Pinus resinosa) durch Sphaeropsis sapinea in sieben Baumschulen im Gebiet der Grossen Seen
Summary
enThe fungus Sphaeropsis sapinea persists on or in stems of asymptomatic red pine (Pinus resinosa) nursery seedlings, and proliferates to cause collar rot and mortality after planting. In the spring of 2002, seven nurseries were surveyed to determine the potential range in frequency of asymptomatic persistence: three operated by Wisconsin Department of Natural Resources (DNR), two by Minnesota DNR, one by Michigan DNR, and one by USDA Forest Service (in Michigan). At each nursery five groups of 20 asymptomatic red pine seedlings were collected near an inoculum source (red pine windbreak), if present, and five groups of 20 asymptomatic seedlings were collected away from such a source (1400 seedlings total). A segment of the lower stem/root collar from each seedling was surface disinfested and incubated on tannic acid agar. Transfers were made from resulting colonies and the pathogen identified from pycnidia and conidia produced in culture. The pathogen was identified from asymptomatic seedlings collected in all Wisconsin and Minnesota nurseries, but was never detected from seedlings from the Michigan DNR or USDA Forest Service nurseries. Frequencies of detection were greater (as high as 88%) from asymptomatic seedlings near red pine windbreaks including diseased trees than from seedlings distant from such windbreaks. A subset of isolates from asymptomatic seedlings was characterized using inter-simple sequence repeat–polymerase chain reaction analysis. Most isolates were the A group of S. sapinea, but B group isolates (recently named Diplodia scrobiculata) were also obtained from one nursery. One Minnesota nursery was more extensively sampled in 2003, with 17–44 groups of five asymptomatic red pine seedlings collected in four separate fields (525 seedlings total). The mean frequency of detection of the pathogen in these four fields ranged from 40 to 71%. Persistence of S. sapinea on or in asymptomatic seedlings continues to be problematic, not only because of the potential for subsequent seedling mortality, but also as a means by which a pathogen may be widely distributed.
Résumé
frLe champignon Sphaeropsis sapinea se maintient de façon asymptomatique sur ou dans les pousses des semis de Pin rouge (Pinus resinosa) en pépinières, et prolifère après plantation, causant des nécroses de collet et des mortalités. Au cours du printemps 2002, des prospections ont été réalisées dans sept pépinières pour déterminer l'amplitude potentielle de fréquence de la présence asymptomatique: trois pépinières gérées par le Wisconsin Department of Natural Resources (DNR), deux par le Minnesota DNR, une par le Michigan DNR, et une par l'U.S.D.A. Forest Service (au Michigan). Dans chaque pépinière, cinq groupes de vingt semis asymptomatiques de Pin rouge ont été récoltés d'une part à proximité d'une source d'inoculum (volis de Pin rouge), quand il y en avait, d'autre part à l’écart de la source (1400 semis au total). Un isolement èté réalisé pour chaque semis à partir d'un segment de la partie basale de la tige/collet, désinfecté superficiellement et incubé sur un milieu gélosé avec de l'acide tannique. Les colonies résultantes ont été repiquées et le pathogène identifiéà partir des pycnides et conidies produites en culture. Le pathogène a été détectéà partir de semis asymptomatiques pour toutes les pépinières du Wisconsin et du Minnesota, mais jamais en provenance des pépinières du Michigan (gérées par le DNR ou l'USDA Forest Service). La fréquence de détection est plus forte (jusqu’à 88%) pour les semis asymptomatiques situés à proximité de volis, incluant des arbres malades, que pour les semis plus éloignés de ces arbres. Un échantillon d'isolats de semis asymptomatiques a été caractérisé par ISSR-PCR. La plupart des isolats appartiennent au groupe A de Sphaerospis sapinea mais des isolats du groupe B (récemment renomméDiplodia scrobiculata) ont également été obtenus à partir d'une pépinière. Une pépinière du Minnesota a fait l'objet d'une prospection plus intensive en 2003, avec récolte de 17 à 44 groupes de cinq semis asymptomatiques dans quatre champs distincts (525 semis au total). La fréquence moyenne de détection du pathogène dans ces quatre champs varie entre 40 et 71%. Le maintien asymptomatique de S. sapinea sur ou dans les semis est un problème non seulement du fait des mortalités potentielles qui peuvent suivre mais aussi parce qu'il peut favoriser une large dissémination du pathogène.
Zusammenfassung
deSphaeropsis sapineaüberdauert endo- und epiphytisch in symptomlosen Stämmen von Pinus resinosa Sämlingen in Baumschulen. Der Pilz breitet sich von dort weiter aus und verursacht eine Wurzelhalsfäule, an der die Pflanzen eingehen. Die Häufigkeit der symptomlosen Besiedlung wurde im Frühling 2002 in sieben Baumschulen erhoben: drei Baumschulen des Wisconsin Department of Natural Resources (DNR), zwei Baumschulen des Minnesota DNR und je eine des Michigan DNR und des U.S.D.A. Forest Service (in Michigan). In jeder Baumschule wurden fünf Gruppen von je 20 symptomlosen Rotkiefer-Sämlingen in der Nähe einer Inokulumsquelle (Windwurffläche mit Rotkiefer), falls eine solche vorhanden war, und fünf gleich grosse Gruppen von Sämlingen, die entfernt von dieser Quelle wuchsen, gesammelt (insgesamt 1400 Sämlinge). Von jedem Sämling wurde ein Segment aus dem Übergangsbereich Stamm-Wurzelhals entnommen, oberflächendesinfiziert und auf Tanninagar inkubiert. Von den auswachsenden Myzelien wurden Reinkulturen angelegt und das Pathogen anhand der in diesen Kulturen gebildeten Pyknidien und Konidien identifiziert. Das Pathogen konnte in symptomlosen Sämlingen sämtlicher Baumschulen von Wisconsin und Minnesota festgestellt werden, war aber nie in oder auf den Pflanzen aus den Baumschulen des Michigan DNR oder des U.S.D.A. Forest Service vorhanden. Die Häufigkeit des Pilzes war in Pflanzen nahe von Windwurfflächen mit an S. sapinea erkrankten Rotkiefern grösser (bis 88%) als in Pflanzen, die entfernt von solchen Windwurfflächen wuchsen. Ein Teil der Isolate von den symptomlosen Sämlingen wurde mittels ISSR-PCR charakterisiert. Die meisten Isolate gehörten zur A Gruppe von S. sapinea, nur in einer Baumschule wurden auch Isolate der Gruppe B (die kürzlich als Diplodia scrobiculata beschrieben wurde) nachgewiesen. Eine ausgedehntere Untersuchung wurde in einer der beiden Baumschulen in Minnesota durchgeführt. Es wurden 17 bis 44 Gruppen von je 5 symptomlosen Sämlingen in vier verschiedenen Feldern gesammelt (insgesamt 525 Sämlinge). Die durchschnittliche Häufigkeit des Pathogens in diesen Feldern betrug zwischen 40 und 71%. Die Überdauerung von S. sapinea in oder auf symptomlosen Sämlingen bleibt ein Problem, nicht nur wegen der Sämlingsmortalität sondern auch wegen der Ausbreitung des Pathogens mit ,,gesunden’’ Baumschulpflanzen.
1 Introduction
Sphaeropsis sapinea (Fr. : Fr.) Dyko and Sutton is among the most common and widespread pathogens of pines and other conifers (Punithalingam and Waterston 1970; Farr et al. 1989). Sphaeropsis shoot blight, canker, and crown wilt diseases have caused extensive damage to native or introduced pines in nurseries, plantations, shelterbelts and forests (Peterson and Wysong 1968; Chou 1976, 1987; Currie and Toes 1978; Palmer and Nicholls 1985; Nicholls and Ostry 1990; Zwolinski et al. 1990; Swart and Wingfield 1991). In addition, rapid death of many recently planted seedlings and established saplings in red pine (Pinus resinosa Aiton) plantations in Wisconsin has been associated with Sphaeropsis collar rot (Stanosz and Cummings Carlson 1996).
Investigations of the aetiology of collar rot led to the understanding of S. sapinea as a latent pathogen (sensuMussell 1980). Sampling at two Wisconsin nurseries in 1994 revealed that virulent strains of S. sapinea persist on or in red pine seedlings in the absence of gross symptoms (Stanosz et al. 1997). In greenhouse experiments it was subsequently demonstrated that host water stress can stimulate proliferation of the pathogen in the lower stem or root collar to rapidly girdle stems and kill young trees (Stanosz et al. 2001).
Two forms of S. sapineasensu lato are encountered in the Great Lakes region of the US. Originally proposed to differ on the basis of morphological characteristics (Palmer et al. 1987), A and B groups can be differentiated based on analysis of a variety of molecular markers, including isozymes, random amplified polymorphic DNA and inter-simple sequence repeat (ISSR) markers, and sequences of the internal transcribed spacer and 5.8S rDNA, microsatellite loci, and protein coding genes (Stanosz et al. 1999; Zhou et al. 2001; Zhou and Stanosz 2001; De Wet et al. 2003). Differences led to the recent assignment of the name Diplodia scrobiculata J. de Wet, B. Slippers, and M. J. Wingfield to the B group by De Wet et al. (2003). Isolates obtained from asymptomatic red pine nursery seedlings in Wisconsin have been characterized as A group (Stanosz et al. 1997).
Recent episodes of collar rot in young plantations in Wisconsin, Michigan, and Minnesota prompted additional surveys for asymptomatic persistence of S. sapinea on or in red pine seedlings in nurseries. Two Wisconsin nurseries that were previously sampled were resampled, and five additional nurseries in Michigan, Minnesota, and Wisconsin also were sampled. Seedlings were culturally assayed for the pathogen, and isolates were characterized by analysis of ISSR-PCR markers. In addition, the possible influences of proximity to a primary inoculum source and incidence of symptomatic seedlings on frequency of asymptomatic persistence were examined.
2 Materials and methods
2.1 2002 Survey
Seven nurseries (Table 1) operated by state or federal agencies that produce bareroot red pine seedlings for planting on public or private lands were surveyed in early spring 2002, before seedlings broke dormancy. Seedlings had arisen from seed sown in the fall of 1999 and had completed two seasons of growth at all nurseries except the Toumey Nursery, where half the surveyed seedlings were from seed sown in the spring of 1999 and half were from seed sown in the spring of 2000.
| Agency2 | Nursery | Location (lat., long.) | Inoculum3 pressure | Seedlings symptomatic (%) (dead, or alive but blighted) | Symptomatic seedlings positive for S. sapinea (no. positive/no. examined) | Seedlings asymptomatic but positive for S. sapinea (%) | ||
|---|---|---|---|---|---|---|---|---|
| Mean4,5 | Range | Mean5,6 | Range | |||||
| WI DNR | Griffith (f)7 | WI Rapids, WI (44.39°N, 89.83°W) | High | 21a | 14–34 | 9/10 | 35a | 20–70 |
| Low | 1A* | 0–4 | 3A* | 0–10 | ||||
| Hayward (f) | Hayward, WI (46.01°N, 91.48°W) | High | 26ab | 20–31 | 9/10 | 30a | 20–35 | |
| Low | 12B* | 5–16 | 24B | 15–40 | ||||
| Wilson (f) | Boscobel, WI (43.14°N, 90.70°W) | High | 43bc | 25–59 | 10/10 | 63b | 35–75 | |
| Low | 8AB* | 0–14 | 8AB* | 0–25 | ||||
| MN DNR | Badoura | Badoura, MN (46.85°N, 94.72°W) | High | 58c | 41–72 | 10/10 | 88c | 75–95 |
| Low | 4AB* | 1–7 | 19AB* | 5–50 | ||||
| General Andrews | Willow River, MN (46.32°N, 92.84°W) | – | 4 | 2–6 | 10/10 | 26 | 5–40 | |
| – | 4 | 1–6 | 20 | 10–35 | ||||
| MI DNR | Wyman | Manistique, MI (45.96°N, 86.25°W) | – | 3 | 0–7 | 0/10 | 0 | 0–0 |
| – | 1 | 0–2 | 0 | 0–0 | ||||
| USDA, FS | Toumey (f) | Watersmeet, MI (46.25°N, 89.21°W) | – | 1 | 0–4 | 0/4 | 0 | 0–0 |
| – | 0 | 0–1 | 0 | 0–0 | ||||
- 1Seedlings had completed two seasons of growth, except at Toumey where seedlings in one of the two areas had completed a third season of growth (spring-sown 3 years earlier).
- 2Departments of Natural Resources of Wisconsin (WI), Minnesota (MN), and Michigan (MI), and the US Department of Agriculture, Forest Service.
- 3Two areas within each of the Griffith, Hayward, Wilson, and Badoura nurseries were selected to differ in proximity to a primary inoculum source (red pine windbreak) and preliminary visual estimate of incidence of symptoms. At General Andrews, Wyman, and Toumey nurseries, neither such a difference in proximity to a primary inoculum source nor incidence of symptoms were apparent and the two areas sampled were not considered to represent differences in exposure to inoculum.
- 4Mean and range (to nearest %) for five plots; each plot consisted of all seedlings (usually 60–90) within a 1-m-long segment of an interior row.
- 5Data from Griffith, Hayward, Wilson, and Badoura nurseries only were analysed after applying the arcsine of the square root transformation to the proportions. Values (within a column) for high inoculum pressure that are followed by the same lower case letter are not significantly different at p < 0.05, and values (within a column) for low inoculum pressure that are followed by the same upper case letter are not significantly different at p < 0.05 using Tukey simultaneous Tests. Asterisks indicate differences at p < 0.05 between values for high and low inoculum pressure within each nursery.
- 6Mean and range (to nearest %) for five plots; 20 asymptomatic seedlings per plot were assayed.
- 7The ‘f’ indicates that fungicide was applied during each year of seedling production (benomyl at Griffith, Hayward, and Wilson; chlorothalonil at Toumey).
Application of fungicides in spring and early summer when expanding shoots are susceptible can reduce incidence of Sphaeropsis shoot blight on red pine seedlings (Palmer et al. 1986; Stanosz and Smith 1996). Fungicide use in spring and early summer during the production of the seedlings sampled for these surveys differed among nurseries. Benomyl had been applied in each year of production at the Griffith, Hayward, and Wilson nurseries, and chlorothalonil at the Toumey Nursery. No fungicides were routinely applied during this portion of the growing season at the Badoura, General Andrews, and Wyman nurseries.
Red pine windbreaks including diseased trees that can serve as a source of S. sapinea inoculum (Palmer et al. 1988) were present at the Griffith, Hayward, Wilson, and Badoura nurseries and symptoms were common on seedlings in beds near these windbreaks. At each of these four nurseries, seedlings were surveyed in two areas: in beds adjacent to windbreaks (high inoculum pressure) and also in beds distant from these windbreaks (low inoculum pressure). At one additional nursery (General Andrews Nursery), although a red pine windbreak was present, symptoms were not observed on windbreak trees and did not appear to be common on seedlings in beds near this windbreak. At the Wyman and Toumey nurseries, red pines were not present (or were rare) in windbreaks, and again symptoms did not appear to be common on seedlings in beds near windbreaks. At the General Andrews, Wyman, and Toumey nurseries seedlings were surveyed both in beds adjacent to a windbreak and in beds distant from a windbreak, but these areas were not considered to represent differences in exposure to inoculum.
Five plots in which seedlings were visually examined to determine the incidence of shoot blight symptoms including mortality were established at each of two survey areas (adjacent to or distant from a windbreak) in each nursery. Each plot consisted of all seedlings within a 1-m-long segment of an interior row; these were counted (usually 60–90 seedlings) and the numbers symptomatic or asymptomatic were recorded. Twenty arbitrarily selected asymptomatic seedlings from within each surveyed metre of row were then cut near the soil line, bagged, and placed in a cooler on ice until return to the laboratory, where seedlings were stored at 4°C for up to 3 days before processing. In addition, up to 10 (if available) symptomatic seedlings from each nursery were bagged and later examined for the presence of S. sapinea as indicated by characteristics of pycnidia and conidia (Punithalingam and Waterston 1970).
Each of the 1400 collected asymptomatic seedlings (100 per area surveyed per nursery) was culturally assayed for the presence of S. sapinea. A segment approximately 5–6 cm long of the lower stem and/or root collar of each seedling was cut, the needles were removed, and it was then surface disinfested by immersion for 30 s in 95% ethanol, and then two immersions for 2 min each in 1.05% NaClO plus two drops Tween-80 per litre. Stem segments were then placed on tannic acid agar (Blodgett et al. 2003) in Petri dishes and incubated in the dark at ambient laboratory temperatures. Transfers were made from the margins of subsequently produced colonies to water agar with autoclaved red pine needles in sterile glass slants that were incubated for at least 1 month at ambient laboratory temperature and light. Pycnidia produced on or in the agar or pine needles were examined and S. sapinea was identified on the basis of conidial characteristics (Punithalingam and Waterston 1970).
Ten arbitrarily selected isolates obtained from asymptomatic seedlings from each of the five nurseries where S. sapinea was detected were characterized by ISSR-PCR fingerprint analysis. Briefly, single conidial isolates were grown in potato dextrose broth and DNA was extracted as described by Smith and Stanosz (1995). A PCR reaction was performed using primer 5′-HYH(GT)7 (Zhou et al. 2001) using conditions detailed by Smith et al. (2003). DNA fragments were visualized in a 0.7% agarose gel stained with ethidium bromide and photographed. Isolates were characterized as either the A or B group of S. sapinea by the distinctive fingerprint patterns this primer yields (Zhou et al. 2001).
2.2 2003 Survey
In response to concern about the potential for distribution of the pathogen on or in asymptomatic seedlings from the Badoura Nursery and subsequent seedling mortality, a second survey was conducted there on 29 and 30 July 2003. Red pine seedlings were sampled in four large fields in which seedlings were completing their third season of growth and were intended for lifting and shipment in the spring of 2004. Due to the results obtained for the field sampled in 2002, seedlings were destroyed and the field was not included in the 2003 survey. A variable number of plots (Table 2), depending on the number and length of the beds of red pine seedlings present, were established at regular intervals across each field (105 plots total). Each plot consisted of all seedlings within a 1-m-long segment of an interior row; these were counted (usually 60–90 seedlings) and the numbers symptomatic or asymptomatic were recorded. Five arbitrarily selected asymptomatic seedlings from within each surveyed metre of row were then cut near the soil line, bagged, and placed in a cooler on ice until return to the laboratory, where seedlings were stored at 4°C for up to 3 days before processing. Each of the 525 collected asymptomatic seedlings was culturally assayed for the presence of S. sapinea as described above for the 2002 survey.
| Field | No. plots | Seedlings symptomatic (%) (dead, or alive but blighted) | Seedlings asymptomatic but positive for S. sapinea (%) | ||
|---|---|---|---|---|---|
| Mean2,3 | Range | Mean3,4 | Range | ||
| B1 | 31 | 14b | 0–27 | 68bc | 0–100 |
| B9 | 13 | 7a | 0–25 | 40a | 0–100 |
| B10 | 17 | 11ab | 0–35 | 47ab | 0–100 |
| D10 | 44 | 14b | 0–36 | 71c | 0–100 |
- 1Seedlings were examined in late July of their third season of growth.
- 2Mean and range (to nearest %) for the number of plots indicated; each plot was an approximate 1-m-long portion of an interior row.
- 3Data were analysed after applying the arcsine of the square root transformation to the proportions. Values (within a column) that are followed by the same letter are not significantly different at p < 0.05 as indicated by Tukey simultaneous tests.
- 4Mean and range (to nearest %) for the number of plots indicated; five asymptomatic seedlings per plot were assayed.
2.3 Statistical analyses
Data from the four nurseries at which seedlings were examined in areas both adjacent to and distant from inoculum sources in 2002 were analysed by two-way analyses of variance to test for effects of nursery, inoculum pressure, and their interaction on incidence of symptomatic seedlings and frequency of detection of S. sapinea from asymptomatic seedlings. For data from these same four nurseries, one-way analyses of variance and Tukey simultaneous tests were then used to examine effects of nursery at high and low inoculum pressures (separately) and to examine effects of high and low inoculum pressures at each nursery (separately). One-way analyses of variance were used to test effect of field on occurrence of symptomatic seedlings and frequency of detection of S. sapinea from asymptomatic seedlings at Badoura in 2003, and differences among mean values again were determined using Tukey simultaneous tests. Percentage data were converted to proportions and the arcsine of the square root transformation was applied before performing these analyses of variance. For the data from these same four nurseries in 2002 and the Badoura Nursery in 2003, relationships between percentages of symptomatic seedlings and asymptomatic seedlings positive for S. sapinea were examined by calculation of the Pearson product–moment correlation coefficient (r). All analyses were performed using Minitab for Windows version 14 (Minitab Inc., State College, PA, USA).
3 Results
3.1 2002 Survey
Incidence of symptomatic seedlings varied widely among and within the nurseries (Table 1). For the four nurseries in which seedlings were sampled in beds both adjacent to (high inoculum pressure) and at a distance from (low inoculum pressure) red pine windbreaks including diseased trees, two-way analysis of variance of the transformed data indicated effects of nursery (p < 0.001), inoculum pressure (p < 0.001), and their interaction (p < 0.001) on incidence of symptomatic seedlings. Subsequent analyses of data for each level of inoculum pressure separately indicated differences among nurseries (at values of p < 0.05). For example, at high inoculum pressure 58% of seedlings at Badoura were symptomatic, compared with only 21% at Griffith. Similarly, at low inoculum pressure 12% of seedlings at Wilson were symptomatic, compared with only 1% at Griffith. However, one-way analyses of variance for each nursery separately indicated that mean values for incidence of symptomatic seedlings in beds adjacent to these windbreaks always were greater than in respective beds at a distance (values of p < 0.05). For example, mean values for incidence of symptomatic seedlings at Badoura Nursery in Minnesota were 58% at high inoculum pressure and 4% at low inoculum pressure. Examination of the interaction plot confirmed that the significance of the interaction of nursery and inoculum pressure on frequency of symptomatic seedlings resulted from differences in the relative effect of inoculum pressure at the different nurseries. For example, compared with 21 and 1% at Griffith, the frequencies of symptomatic seedlings at high and low inoculum pressures at Hayward were 26 and 12% respectively. Percentages of symptomatic seedlings at the remaining three nurseries (without red pine windbreaks or lacking symptomatic trees in red pine windbreaks) were very low (≤4%) (Table 1).
Detection of S. sapinea on the collected symptomatic seedlings differed among the nurseries (Table 1). Characteristic pycnidia and conidia were obtained from nine of 10 or 10 of 10 seedlings examined from the Griffith, Hayward, and Wilson nurseries in Wisconsin and the Badoura and General Andrews nurseries in Minnesota. In contrast, signs of the pathogen were not found on the seedlings from the Wyman and Toumey nurseries in Michigan.
Frequency of identification of S. sapinea from asymptomatic seedlings also varied widely among and within the nurseries (Table 1). For the four nurseries in which seedlings were sampled in beds both adjacent to (high inoculum pressure) and at a distance from (low inoculum pressure) red pine windbreaks including diseased trees, two-way analysis of variance of the transformed data indicated effects of nursery (p < 0.001), inoculum pressure (p < 0.001), and their interaction (p < 0.001) on frequency of asymptomatic seedlings positive for S. sapinea. Subsequent analyses of data for each level of inoculum pressure separately indicated differences among nurseries. For example, at high inoculum pressure 88% of asymptomatic seedlings at Badoura were positive for S. sapinea, compared with only 30% at Hayward. Similarly, at low inoculum pressure 24% of asymptomatic seedlings at Hayward were positive for S. sapinea, compared with only 3% at Griffith. However, one-way analyses of variance for each nursery separately indicated that mean values for frequency of asymptomatic seedlings positive for S. sapinea in beds adjacent to these windbreaks were greater than in respective beds at a distance in three of these four nurseries (values of p < 0.05). For example, mean values for frequency of asymptomatic seedlings positive for S. sapinea at Griffith were 35% at high inoculum pressure and 3% at low inoculum pressure. Examination of the interaction plot confirmed that the significance of the interaction of nursery and inoculum pressure on frequency of asymptomatic seedlings positive for S. sapinea resulted from differences in the relative effect of inoculum pressure at the different nurseries. For example, compared with 35 and 3% at Griffith, the frequencies of asymptomatic seedlings positive for S. sapinea at high and low inoculum pressure at Hayward were 30 and 24% respectively. Sphaeropsis sapinea was never detected from asymptomatic seedlings at the Wyman and Toumey nurseries in Michigan. However, it was obtained from ≥20% of asymptomatic seedlings at the General Andrews Nursery, where a red pine windbreak was present but symptoms were not observed on windbreak trees and were not common on seedlings (Table 1).
Incidence of symptomatic seedlings was positively correlated with frequency of detection of S. sapinea from asymptomatic seedlings (Fig. 1a). The Pearson product–moment correlation coefficient was 0.892 (p < 0.001). At relatively low incidences of symptomatic seedlings (e.g. from approximately 15–20%), however, frequency of detection of the pathogen from asymptomatic seedlings varied widely (e.g. from 0 to 40%).
Relationship between incidence of symptomatic seedlings in plots in red pine nursery beds and frequency of identification of Sphaeropsis sapinea from asymptomatic seedlings from those plots: (a) data from Griffith, Hayward, Wilson, and Badoura nurseries sampled in 2002 (r = 0.892, p < 0.001); (b) data from Badoura Nursery sampled in 2003 (r = 0.375, p < 0.001). In both years, plots in which incidence of symptomatic seedlings was determined consisted of all seedlings within a 1-m-long segment of an interior row; frequency of asymptomatic seedlings positive for S. sapinea was determined by cultural assay of 20 (2002) or five (2003) asymptomatic seedlings from each plot
The results of the ISSR-PCR characterization of isolates varied among nurseries. All 40 isolates from the Griffith, Hayward, Wilson, and Badoura nurseries were characterized as A group. In contrast, seven of 10 isolates from the General Andrews Nursery were characterized as B group, with the remaining three isolates from this nursery characterized as A group.
3.2 2003 Survey
For the four fields sampled in 2003, mean incidence of symptomatic seedlings varied from 7 to 14% and mean frequency of detection of S. sapinea from asymptomatic seedlings varied from 40 to 71% (Table 2). Analyses of variance indicated significant effect of field on each of these variables (values of p < 0.005). Incidence of symptomatic seedlings was positively correlated with frequency of detection of S. sapinea from asymptomatic seedlings (Fig. 1b). The Pearson product–moment correlation coefficient was 0.375 (p < 0.001). At even very low incidences of symptomatic seedlings (e.g. 0–15%), however, frequency of detection of the pathogen from asymptomatic seedlings varied widely (e.g. from 0 to 100%).
4 Discussion
Occurrence of shoot blight at five of seven nurseries and the asymptomatic persistence of S. sapinea on or in seedlings at these nurseries indicates a continuing threat to red pine health. Symptomatic seedlings are not acceptable for sale and are a source of economic loss to the nursery. Asymptomatic persistence of the pathogen presents a risk of subsequent seedling mortality, as well as providing a means of long-distance dissemination of the pathogen. The lack of detection on the seedlings from the Toumey and Wyman nurseries in Michigan, however, indicates that asymptomatic persistence of S. sapinea on or in red pine nursery stock is not inevitable. Current knowledge of the biology of the S. sapinea–red pine interaction should prompt changes in management to reduce damage both in nursery production and after outplanting.
The frequencies of symptomatic seedlings observed in proximity to red pine windbreaks including diseased trees, 21–58% in the current surveys, can be compared with those mentioned in past studies of Sphaeropsis shoot blight. For example, Palmer and Nicholls (1985) reported losses of approximately 16% of 2- and 3-year-old seedlings at the Wilson Nursery in 1982. In non-treated control plots of fungicide trials at the same nursery (Palmer et al. 1986), approximately 42% of seedlings were diseased in 1982 and 1983, and approximately 74% in 1981. But in the current surveys, frequencies of symptomatic seedlings in beds that had been treated with benomyl near windbreaks in Griffith, Hayward, and Wilson nurseries (21, 26 and 43%, respectively) far exceed those reported for benomyl-treated seedlings in past fungicide trials. Palmer et al. (1986) reported incidence of only 2.7% when seedlings were treated with benomyl each of 2 years of growth at the Wilson Nursery. In proximity to a red pine windbreak inoculum source, only approximately 10% of 2-year-old seedlings in plots treated with benomyl were affected by shoot blight at the Griffith Nursery (Stanosz and Smith 1996). Thus, our current results from the Griffith, Hayward, and Wilson nurseries indicate that benomyl application may not prevent relatively high incidence of Sphaeropsis shoot blight under high inoculum pressure.
The frequencies of detection of S. sapinea from asymptomatic seedlings in the current surveys also can be compared with those reported previously. The pathogen was detected from 21 and 34%, respectively, of asymptomatic red pine seedlings sampled in 1994 from the Griffith and Wilson nurseries (Stanosz et al., 1997), figures similar to those obtained for seedlings near windbreaks in Griffith and Hayward nurseries in 2002. However, the two- to three-fold greater frequencies of detection from similar beds in the Wilson and Badoura nurseries in 2002, and from fields sampled at Badoura in 2003, indicate that S. sapinea may persist on or in a majority of asymptomatic seedlings when inoculum is abundant. In addition to spores disseminated from diseased windbreak trees, sporulation of S. sapinea on symptomatic seedlings that were common in these beds is a likely source of additional inoculum that may have contributed to the observed high frequencies of asymptomatic persistence. Finally, consistent with results from the 1994, application of benomyl to sampled beds at the Griffith, Hayward, and Wilson nurseries did not eliminate the asymptomatic persistence of S. sapinea on or in seedlings. The ability to inhibit development of this asymptomatic relationship should be considered, in addition to minimization of disease symptoms in the nursery, in future testing of fungicides to prevent damage from S. sapinea.
Prediction of the frequency of persistence of S. sapinea on or in asymptomatic seedlings based on more easily obtained data for the incidence of blighted seedlings might allow managers to estimate the risk of development of Sphaeropsis collar rot and mortality following outplanting. Knowledge of high frequencies of asymptomatic persistence might justify destruction of seedlings rather than sale. In Griffith, Hayward, Wilson, and Badoura nurseries in 2002, high frequencies of symptomatic seedlings were indicative of high frequencies of asymptomatic detection of the pathogen. This correlation is consistent with the observation of Flowers et al. (2001) who cultured S. sapinea from asymptomatic shoots of Austrian pine (P. nigra Arnold). Asymptomatic shoots from trees with ≥25% blighted shoot tips more frequently yielded the fungus than asymptomatic shoots from trees with <20% blighted shoot tips. But regardless of the statistically significant correlations obtained for the data from Griffith, Hayward, Wilson, and Badoura nurseries in 2002 and Badoura Nursery in 2003, accuracy of prediction is limited. In these nurseries there were substantial ranges in the percentages of asymptomatic seedlings from which S. sapinea was detected even when relatively low percentages of seedlings were symptomatic. Thus, low frequencies of symptomatic seedlings cannot be relied upon to indicate low frequencies of asymptomatic persistence.
The detection of S. sapinea from approximately one of four asymptomatic seedlings from the General Andrews nursery even though blighted or dead seedlings were scarce, also indicates another possible difficulty in attempting to predict asymptomatic persistence. The relative lack of symptomatic seedlings might be explained by the characterization of seven of 10 isolates from this nursery as group B (or D. scrobiculata). Although pathogenic, B group isolates may be less aggressive on red pine than those of the A group. In multiple experiments, red pine seedlings either non-wounded and inoculated with conidia or wounded and inoculated with mycelium developed necrotic needles, resinosis, stem lesions, or dead shoot tips (Blodgett et al. 1997; Blodgett and Stanosz 1997, 1999). But the relative incidence and severity of symptoms resulting from inoculation with B group isolates were low, compared with those resulting from inoculation with A group isolates. For example, Blodgett and Stanosz (1997) reported incidence of symptoms resulting from conidial inoculation of red pine seedling shoot tips to be 18% for B group isolates, compared with 97% for A group isolates. However, our discovery that the B group may also persist on or in red pine nursery seedlings in the absence of symptoms should prompt additional investigation of the interaction of D. scrobiculata and red pine. Palmer (1991) reported obtaining B group isolates from 12 to 32-year-old red pine trees exhibiting cankers and top dieback in Wisconsin and Minnesota. More recently Blodgett and Bonello (2003) reported that inoculation of the bases of Austrian pine stems with B group isolates resulted in much longer cankers than those resulting from shoot tip inoculations. Thus, results of studies cited above in which red pine shoot tip inoculation with B group isolates resulted in relatively low incidence and severity of symptoms may not have reflected the potential for a far more widespread and damaging interaction.
The relatively high frequencies of detection of S. sapinea from asymptomatic seedlings in beds in proximity to windbreaks at Griffith, Hayward, Wilson and Badoura nurseries has provided additional motivation for adopting long-standing (but until recently largely unheeded) recommendations to reduce losses from Sphaeropsis shoot blight. Following studies of the sources of inoculum in nurseries and description of a gradient of decreasing Sphaeropsis shoot blight incidence with increasing distances from windbreaks, Palmer et al. (1988) wrote: ‘… local sources of inoculum are important in epidemics of this disease’ and ‘… inoculum sanitation is an important tool for managing outbreaks of S. sapinea in forest tree nurseries’ and ‘trees infected by S. sapinea should be removed and windbreaks should be converted to nonhost species.’ After discussion of our results and their implications with managers, red pine windbreak removal has been accomplished or is planned for the near future at the Minnesota and Wisconsin nurseries. Replacement of windbreaks may not only reduce or eliminate the need for fungicide application to prevent shoot blight in the nursery, but also reduce or eliminate the subsequent threat of collar rot mortality of outplanted seedlings and dissemination of the pathogen from nurseries to planting sites.
Acknowledgements
The authors acknowledge support from the USDA Forest Service, the Minnesota DNR, and the Wisconsin DNR. We also thank nursery personnel for access and assistance in sampling, and JoAnne Stanosz, Isabel Munck, Maria Newcomb, Kelly Finnegan, Mike LaBissoniere, Rachel Leisso, and Mary Stanosz for help processing seedlings. The helpful comments of the editor and reviewers are also gratefully acknowledged.
