- Email: [email protected]
Horizontal transmission of the Picea glauca foliar endophyte Phialocephala scopiformis CBS 120377
fungal ecology 2 (2009) 98–101
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/funeco
Horizontal transmission of the Picea glauca foliar endophyte Phialocephala scopiformis CBS 120377 J. David MILLERa,*, Hafsa CHERIDa, Mark W. SUMARAHa, Gregory W. ADAMSb a
Ottawa-Carleton Institute of Chemistry, Carleton University, 228 Steacie Building, Ottawa, Ontario K1S 5B6, Canada JD Irving Limited, Sussex Tree Nursery, 181 Aiton Road, Sussex, New Brunswick E4G 2V5, Canada
b
article info
abstract
Article history:
We have studied the presence of the foliar endophtye of Picea glauca (white spruce) Phia-
Received 29 October 2008
locephala scopiformis CBS 120377 and its affect on the growth of Choristoneura fumiferana
Accepted 5 January 2009
(spruce budworm). Here we examine the transmission of this fungus from 50 trees planted
Published online 10 February 2009
in a test field site to 250 P. glauca seedlings planted under the emerging canopies. After 3 y,
Corresponding editor: Kevin Hyde
the endophyte spread to 40 % of these trees (now 20–30 cm) with an average rugulosin (an anti-insect toxin) concentration of 1 mg g 1. All woody plants within 2 m of the test trees
Keywords:
were collected. These were all shown to be negative for P. scopiformis except for some
Choristoneura fumiferana
spruce seedlings that arose from seeds (natural generation). This is positive evidence for
ELISA
the horizontal transmission of P. scopiformis and its apparent specificity to P. glauca under
Endophyte
field conditions.
Horizontal transmission
ª 2009 Elsevier Ltd and The British Mycological Society. All rights reserved.
Picea glauca Rugulosin
Foliar fungal endophytes produce symptomless infections in plant leaves and sometimes the vascular tissue (Arnold 2007, Sa´nchez Ma´rquez et al. 2008, Huang et al. 2008, Hyde & Soytong 2008). The best understood endophyte-plant system is in various grasses that can be colonized by the Balansia endophytes (Sa´nchez Ma´rquez et al. 2007; Wei et al. 2007). The fungus is found inside leaf tissue and produces a number of alkaloids that result in serious health problems for cattle and horses when in pastures but also in various insect pests (Clay & Schardl 2002). Endophyte-positive cultivars used for lawns and golf courses are much more insect tolerant, but also have increased drought and fungal pathogen resistance (Clay & Schardl 2002). We have been studying the foliar endophytes of white spruce (Picea glauca) and their effects on the eastern spruce budworm (Choristoneura fumiferana). Spruce budworm is the most serious forest pest in eastern Canada and the northeast USA. The last major epidemic in Canada
occurred from the 1970’s to the mid 1980’s which resulted in severe defoliation, except where sprayed with pesticide in an effort to keep the forest green (Irland 1980; MacLean et al. 2002). We have demonstrated that P. glauca seedlings can be colonized with strains of the foliar endophyte Phialocephala scopiformis (DAOM 229536, CBS 120377) isolated from surfacesterilized needles of P. glauca on the border of Quebec and New Brunswick. The dominant anti-insect toxin produced by this fungus is rugulosin. The needles of infected seedlings grown in growth chambers, under nursery conditions and in a longterm field test contain rugulosin in concentrations that affect the growth of spruce budworm (Miller et al. 2002, 2008; Sumarah et al. 2008a). We have also demonstrated that spruce budworm growth rates are impaired by rugulosin in infected needles in growth chambers (Miller et al. 2002) and under nursery conditions (Miller et al. 2008).
* Corresponding author. E-mail address: [email protected] (J.D. Miller). 1754-5048/$ – see front matter ª 2009 Elsevier Ltd and The British Mycological Society. All rights reserved. doi:10.1016/j.funeco.2009.01.002
Horizontal transmission of a conifer foliar endophyte
The available ecological literature data, though sparse, indicated that unlike grass endophytes which are mostly vertically transmitted, foliar endophytes of woody plants are transmitted horizontally to seedlings planted immediately under infected trees (Bayman et al. 1998; Frohlich et al. 2000). The Xylaria endophytes of the tropical woody angiosperm Manilkara bidentata (Sapotaceae) were demonstrated to be transmitted horizontally (Bayman et al. 1998). Endophytes of another tropical woody angiosperm, Theobroma cacoa (Malvaceae) have also been shown to be similar. In this case, specialization of its endophytes appears to occur. Factors that might contribute to this include nutrients and secondary metabolites of the leaves of particular plants (Arnold & Herre 2003; Arnold et al. 2003). Data on foliar endophytes of north temperate species of conifers has been largely inferential to date. Miller et al. (2002) examined the needles of a large number of seed-grown white spruce seedlings from a large production nursery and found them free of endophytes (see also Sumarah et al. 2005). Ganley & Newcombe (2006) tested seeds and needles of Pinus monticola (western white pine) for its endophytes and obtained similar results. The strain of P. scopiformis discussed in the present report was isolated from surface-sterilized needles. The fungus has previously been reported to be isolated from apparently healthy roots, branches or twigs of spruce species in the north temperate region (Kowalski & Kehr 1995; Ahlich & Sieber 1996; Barklund & Kowalski 1996). From limited field evidence, its transmission in white spruce was horizontal (Sumarah et al. 2008a). The purpose of this report is to expand these studies and examine the transmission of P. scopiformis to white spruce seedlings, and to look for colonization in adjacent woody plants other than white spruce.
Materials and methods Seedlings Descriptions of the trees and inoculation methods used in these experiments are given in Sumarah et al. (2005, 2008a). Briefly, in early September 2003, 300 (some of the original 330 positives died) endophyte/toxin-positive trees were planted at 2 m 2 m spacing in a test field site ca. 30 km from Sussex, NB, Canada. One year later in July 2004, 250 (15 m old) un-inoculated seedlings were obtained as previously described from the JD Irving, Limited genetic improvement program. Five of these seedlings (20–30 mm tall) were planted around each of 50 randomly selected trees from the 300 planted on this field site. The seedlings were planted in close proximity to the infected trees (50 cm away from the stems). Following that, fibreglass screens comprising an area of 0.25 m2 were placed around a further 50 test trees to collect cast needles. In October of 2007, 4 y after planting the test trees and 3 y after planting the small seedlings, all ‘‘small seedlings’’ were collected (244). At this time, they were all under or immediately beside the test tree (~2 m tall) and were typically 20–30 cm tall. In addition all other woody plants from natural regeneration within 2 m of each infected tree were collected. This included Abies balsamea (17 plants), Pinus strobes (18 plants), Picea rubens (11 plants), 34 unidentifiable (due to
99
the age of the tree) Picea species (likely mostly white spruce), P. glauca (eight plants), 20 Salix species, eleven Betula papyrifera plants, five Viburnum cassinoides plants and three Tsuga canadensis plants, as well as cast needles from the screens. These trees/shrubs were 20–30 cm tall. All samples were frozen, freeze-dried and ground to a fine powder (Sumarah et al. 2008a).
Analysis An ELISA (Enzyme-Linked ImmunoSorbent Assay) analysis was carried out to determine the amount of P. scopiformis in the needles (or leaves). Analysis was performed using a polyclonal antibody test that is specific and sensitive to P. scopiformis. The method limit of detection (LOD) for cell mass was 60 ng g 1 (i.e. 60 ppb) and the limit of quantification (LOQ), i.e. a positive, was 120 ng g 1 dry weight of needle (Sumarah et al. 2005, 2008a) Rugulosin concentration was determined by High Performance Liquid Chromatography with a Diode Array Detector. Briefly, 300 mg of freeze-dried, ground needle was extracted in ice-cold petroleum ether. The suspension was filtered and the extract discarded. The needles were then re-extracted with 10 ml of chloroform. The chloroform extract was washed with 5 % NaHCO3. The first chloroform layer was discarded while the aqueous fraction was acidified to pH 3 and extracted with a further 10 ml of chloroform. The chloroform layer was removed and dried in an amber vial under nitrogen. The dried extracts were re-dissolved in acetonitrile. Analysis was done with an 1100 series Agilent Technologies HPLC-DAD with a 250 4.6 RP column with a gradient elution. Samples were analyzed at 389 nm, the maximum UV/VIS absorption for rugulosin and peak identity was confirmed by full spectrum data from the diode array detector. The LOD and LOQ for rugulosin were both 150 ng g 1 (150 ppb; Sumarah et al. 2008a). For statistical purposes, values negative by ELISA and below the analytical detection limit were entered at half the detection limit for rugulosin (as is normal for exposure characterization). Needles that were negative for rugulosin but positive by ELISA were entered at the detection limit because there was positive evidence for the presence of the fungus in the needle.
Results and discussion Of the small seedlings planted immediately beside and currently underneath the infected trees, approximately 40 % were infected, supporting the first limited observation made at 2 y post planting (six small trees; Sumarah et al. 2008a). The arithmetic mean concentration of the positives was 1.1 mg g 1 with a geometric mean of 0.6 mg g 1 (Fig 1). This is similar to data from previous studies (Sumarah et al. 2005, 2008a; Miller et al. 2008). Biomass of the fungus was present in high concentrations in the cast needles but no toxin was detected, which was also similar to samples collected over two previous years (Sumarah et al. 2008a). P. scopiformis was not detected in non-target plants. Of the 53 spruce seedlings collected, five (9 %) were positive for Picea scopiformis (DAOM 229536, CBS 120377) or a very closely related
100
J.D. Miller et al.
0.9
Consistent with the body of evidence on the specificity of foliar endophytes of woody plants, all of the non-target woody plants were negative for the presence of the fungus under field conditions.
0.8 0.7
log ug/g
0.6
Acknowledgements
0.5
This study was supported by the Atlantic Innovation Fund, JD Irving, Limited and the National Research Council of Canada (IRAP program). HC thanks NSERC for a summer fellowship during this project. We thank Greg Slack, Luke Johnson and Madura Sornarajah for technical expertise as well as Ms. Pat MacDonald, JD Irving, Limited for collecting and identifying the non-target plants. Based on a sequence done in our lab, Dr. Christoph Gruenig (Swiss Federal Institute of Technology) provided the identification of the fungus studied with thanks.
0.4 0.3 0.2 0.1 0.0
RUGULOSIN
references Fig 1 – Rugulosin concentrations in 244 seedlings of white spruce; the length of each box shows where the central 50 % of the values fall, with the edges at the first and third quartiles.
species. Cross-reactivity to very similar strains is possible but is not material between other endophytes (Sumarah et al. 2005). Extensive tests have shown that seedlings leaving production nurseries are free of endophytes (Miller et al. 2002). The research effort that began in 1983 (Miller et al. 1985; Miller 1986; Johnson & Whitney 1989, 1992) was an attempt to understand the ecology and biodiversity of the endophyte population in Maine, New Brunswick, eastern Quebec and Nova Scotia. These and the allied secondary metabolite studies since the mid 1990’s (see Sumarah et al. 2008b) indicated that as reforestation began in 1958 there has been a gradual erosion of the genetic diversity of the foliar endophytes (Miller et al. 2008). This study, when considered with previous work on the absence of endophytes in nursery stock (Miller et al. 2002) and the experimental transmission of test strains (Sumarah et al. 2005) confirms the horizontal infection process for the foliar endophyte under study. It was known that many of the foliar endophytes in the conifers in the northern hemisphere represented a particular ecological and genetic group of mostly undescribed species (Ganley et al. 2004; Seibert 2007). There is evidence from secondary metabolite and DNA sequence information that some of these might also be aquatic hyphomycetes (Sokolski et al. 2006; Sumarah et al. 2008b). When the needles are cast (usually after 3 y in white spruce), it appears that the endophytes act as early saprobic colonizers and sporulate (Oses et al. 2008; Hyde & Soytong 2008). There is much circumstantial evidence for this in tropical plants (Promputtha et al. 2007) and even grasses (Sa´nchez Ma´rquez et al. 2008). In a plantation situation, it appears that efficient transmission is confined to naturally regenerating seedlings quite close to the infected tree. Further spread of aquatic hyphomycetes endophytes might depend on the adventitious colonization of woody debris.
Ahlich K, Sieber TN, 1996. The profusion of dark septate endophytic fungi in non-ectomycorrhizal fine roots of forest trees and shrubs. New Phytologist 132: 259–270. Arnold AE, Herre EA, 2003. Canopy cover and leaf age affect colonization by tropical fungal endophytes: ecological pattern and process in Theobroma cacoa (Malvaceae). Mycologia 95: 388–398. Arnold AE, Mejia LC, Kyllo D, Rjas EI, Maynard Z, Robbins N, Herre EA, 2003. Fungal endophytes limit pathogen damage in a tropical tree. Proceedings of the National Academy of Sciences of the United States of America 100: 15649–15654. Arnold AE, 2007. Understanding the diversity of foliar endophytic fungi: progress, challenges, and frontiers. Fungal Biology Reviews 21: 51–66. Barklund P, Kowalski T, 1996. Endophytic fungi in branches of Norway spruce with particular reference to Tryblidopsis pinastri. Canadian Journal of Botany 74: 673–678. Bayman P, Angulo-Sandoval P, Baez-Ortiz Z, Lodge DJ, 1998. Distribution and dispersal of Xylaria endophytes in two tree species in Puerto Rico. Mycological Research 102: 944–948. Clay K, Schardl C, 2002. Evolutionary origins and ecological consequences of endophyte symbiosis with grasses. American Naturalist 160: S99–S127. Fro¨hlich J, Hyde KD, Petrini O, 2000. Endophytic fungi associated with palms. Mycological Research 104: 1202–1212. Ganley RJ, Brunsfeld SJ, Newcombe G, 2004. A community of unknown, endophytic fungi in western white pine. Proceedings of the National Academy of Sciences of the United States of America 101: 10107–10112. Ganley RJ, Newcombe G, 2006. Fungal endophytes in seeds and needles of Pinus monticola. Mycological Research 110: 318–327. Huang WY, Cai YZ, Hyde KD, Corke H, Sun M, 2008. Biodiversity of endophytic fungi associated with 29 traditional Chinese medicinal plants. Fungal Diversity 33: 61–75. Hyde KD, Soytong K, 2008. The fungal endophyte dilemma. Fungal Diversity 33: 163–173. Irland LC, 1980. Pulpwood, pesticides, and people. Controlling spruce budworm in northeastern North America. Environmental Management 4: 381–389. Johnson JA, Whitney NJ, 1989. A study of fungal endophytes of needles of balsam fir (Abies balsamea) and red spruce (Piceae rubens) in New Brunswick, Canada, using cultural and electron microscope techniques. Canadian Journal of Botany 67: 3513–3516.
Horizontal transmission of a conifer foliar endophyte
Johnson JA, Whitney NJ, 1992. Isolation of fungal endophytes from black spruce (Picea mariana) dormant buds and needles from New Brunswick, Canada. Canadian Journal of Botany 70: 1754–1757. Kowalski T, Kehr RD, 1995. Two new species of Phialocephala occurring on Picea and Alnus. Canadian Journal of Botany 73: 26–32. MacLean DA, Beaton KP, Porter KB, MacKinnon WB, Budd MG, 2002. Potential wood supply losses to spruce budworm in New Brunswick estimated using the Spruce Budworm Decision Support System. Forestry Chronicle 78: 739–750. Miller JD, 1986. Toxins of endophytic and epiphytic fungi of conifer needles. In: Fokkema, N.J., Van Huevel, J. (Eds), Microbiology of the Phyllosphere. Cambridge University Press, Cambridge, pp. 223–231. Miller JD, Strongman D, Whitney NJ, 1985. Observations on fungi associated with spruce budworm infested balsam fir needles. Canadian Journal Forest Research 15: 896–901. Miller JD, Mackenzie S, Foto M, Adams GW, Findlay JA, 2002. Needles of white spruce inoculated with rugulosin-producing endophytes contain rugulosin reducing spruce budworm growth rate. Mycological Research 106: 471–479. Miller JD, Sumarah MW, Adams GW, 2008. Effect of a rugulosinproducing endophyte in Picea glauca on Choristoneura fumiferana. Journal of Chemical Ecology 34: 362–368. Oses R, Valenzuela S, Freer J, Sanfuentes E, Rodrı´guez J, 2008. Fungal endophytes in xylem of healthy Chilean trees and their possible role in early wood decay. Fungal Diversity 33: 77–86.
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Promputtha I, Lumyong S, Dhanasekaren V, McKenzie EHC, Hyde KD, Jeewon R, 2007. Aphylogenetic evaluation of whether endophytes become saprotrophs at host senescence. Microbial Ecology 53: 579–590. Sa´nchez Ma´rquez S, Bills GF, Zabalgogeazcoa I, 2008. Diversity and structure of the fungal endophytic assemblages from two sympatric coastal grasses. Fungal Diversity 33: 87–100. Seibert TN, 2007. Endophytic fungi in forest trees: are they mutualists? Fungal Biology Reviews 21: 75–89. Sokolski S, Piche Y, Chauvet R, Berube JD, 2006. A fungal endophyte of black spruce (Picea mariana) needles is also an aquatic hyphomycete. Molecular Ecology 15: 1955–1962. Sumarah MW, Miller JD, Adams GW, 2005. Measurement of a rugulosin-producing endophyte in white spruce seedlings. Mycologia 97: 770–776. Sumarah MW, Adams GW, Berghout J, Slack G, Wilson A, Miller JD, 2008a. Spread and persistence of a rugulosinproducing endophyte in white spruce seedlings. Mycological Research 112: 731–736. Sumarah MW, Puniani E, Blackwell BA, Miller JD, 2008b. Characterization of polyketide metabolites from foliar endophytes of Picea glauca. Journal of Natural Products 71: 1393–1398. Wei YK, Gao YB, Zhang X, Su D, Wang YH, Xu H, Lin F, Ren AZ, Chen L, Nie LY, 2007. Distribution and diversity of Epichloe¨/ Neotyphodium fungal endophytes from different populations of Achnatherum sibiricum (Poaceae) in the Inner Mongolia Steppe, China. Fungal Diversity 24: 329–345.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/funeco
Horizontal transmission of the Picea glauca foliar endophyte Phialocephala scopiformis CBS 120377 J. David MILLERa,*, Hafsa CHERIDa, Mark W. SUMARAHa, Gregory W. ADAMSb a
Ottawa-Carleton Institute of Chemistry, Carleton University, 228 Steacie Building, Ottawa, Ontario K1S 5B6, Canada JD Irving Limited, Sussex Tree Nursery, 181 Aiton Road, Sussex, New Brunswick E4G 2V5, Canada
b
article info
abstract
Article history:
We have studied the presence of the foliar endophtye of Picea glauca (white spruce) Phia-
Received 29 October 2008
locephala scopiformis CBS 120377 and its affect on the growth of Choristoneura fumiferana
Accepted 5 January 2009
(spruce budworm). Here we examine the transmission of this fungus from 50 trees planted
Published online 10 February 2009
in a test field site to 250 P. glauca seedlings planted under the emerging canopies. After 3 y,
Corresponding editor: Kevin Hyde
the endophyte spread to 40 % of these trees (now 20–30 cm) with an average rugulosin (an anti-insect toxin) concentration of 1 mg g 1. All woody plants within 2 m of the test trees
Keywords:
were collected. These were all shown to be negative for P. scopiformis except for some
Choristoneura fumiferana
spruce seedlings that arose from seeds (natural generation). This is positive evidence for
ELISA
the horizontal transmission of P. scopiformis and its apparent specificity to P. glauca under
Endophyte
field conditions.
Horizontal transmission
ª 2009 Elsevier Ltd and The British Mycological Society. All rights reserved.
Picea glauca Rugulosin
Foliar fungal endophytes produce symptomless infections in plant leaves and sometimes the vascular tissue (Arnold 2007, Sa´nchez Ma´rquez et al. 2008, Huang et al. 2008, Hyde & Soytong 2008). The best understood endophyte-plant system is in various grasses that can be colonized by the Balansia endophytes (Sa´nchez Ma´rquez et al. 2007; Wei et al. 2007). The fungus is found inside leaf tissue and produces a number of alkaloids that result in serious health problems for cattle and horses when in pastures but also in various insect pests (Clay & Schardl 2002). Endophyte-positive cultivars used for lawns and golf courses are much more insect tolerant, but also have increased drought and fungal pathogen resistance (Clay & Schardl 2002). We have been studying the foliar endophytes of white spruce (Picea glauca) and their effects on the eastern spruce budworm (Choristoneura fumiferana). Spruce budworm is the most serious forest pest in eastern Canada and the northeast USA. The last major epidemic in Canada
occurred from the 1970’s to the mid 1980’s which resulted in severe defoliation, except where sprayed with pesticide in an effort to keep the forest green (Irland 1980; MacLean et al. 2002). We have demonstrated that P. glauca seedlings can be colonized with strains of the foliar endophyte Phialocephala scopiformis (DAOM 229536, CBS 120377) isolated from surfacesterilized needles of P. glauca on the border of Quebec and New Brunswick. The dominant anti-insect toxin produced by this fungus is rugulosin. The needles of infected seedlings grown in growth chambers, under nursery conditions and in a longterm field test contain rugulosin in concentrations that affect the growth of spruce budworm (Miller et al. 2002, 2008; Sumarah et al. 2008a). We have also demonstrated that spruce budworm growth rates are impaired by rugulosin in infected needles in growth chambers (Miller et al. 2002) and under nursery conditions (Miller et al. 2008).
* Corresponding author. E-mail address: [email protected] (J.D. Miller). 1754-5048/$ – see front matter ª 2009 Elsevier Ltd and The British Mycological Society. All rights reserved. doi:10.1016/j.funeco.2009.01.002
Horizontal transmission of a conifer foliar endophyte
The available ecological literature data, though sparse, indicated that unlike grass endophytes which are mostly vertically transmitted, foliar endophytes of woody plants are transmitted horizontally to seedlings planted immediately under infected trees (Bayman et al. 1998; Frohlich et al. 2000). The Xylaria endophytes of the tropical woody angiosperm Manilkara bidentata (Sapotaceae) were demonstrated to be transmitted horizontally (Bayman et al. 1998). Endophytes of another tropical woody angiosperm, Theobroma cacoa (Malvaceae) have also been shown to be similar. In this case, specialization of its endophytes appears to occur. Factors that might contribute to this include nutrients and secondary metabolites of the leaves of particular plants (Arnold & Herre 2003; Arnold et al. 2003). Data on foliar endophytes of north temperate species of conifers has been largely inferential to date. Miller et al. (2002) examined the needles of a large number of seed-grown white spruce seedlings from a large production nursery and found them free of endophytes (see also Sumarah et al. 2005). Ganley & Newcombe (2006) tested seeds and needles of Pinus monticola (western white pine) for its endophytes and obtained similar results. The strain of P. scopiformis discussed in the present report was isolated from surface-sterilized needles. The fungus has previously been reported to be isolated from apparently healthy roots, branches or twigs of spruce species in the north temperate region (Kowalski & Kehr 1995; Ahlich & Sieber 1996; Barklund & Kowalski 1996). From limited field evidence, its transmission in white spruce was horizontal (Sumarah et al. 2008a). The purpose of this report is to expand these studies and examine the transmission of P. scopiformis to white spruce seedlings, and to look for colonization in adjacent woody plants other than white spruce.
Materials and methods Seedlings Descriptions of the trees and inoculation methods used in these experiments are given in Sumarah et al. (2005, 2008a). Briefly, in early September 2003, 300 (some of the original 330 positives died) endophyte/toxin-positive trees were planted at 2 m 2 m spacing in a test field site ca. 30 km from Sussex, NB, Canada. One year later in July 2004, 250 (15 m old) un-inoculated seedlings were obtained as previously described from the JD Irving, Limited genetic improvement program. Five of these seedlings (20–30 mm tall) were planted around each of 50 randomly selected trees from the 300 planted on this field site. The seedlings were planted in close proximity to the infected trees (50 cm away from the stems). Following that, fibreglass screens comprising an area of 0.25 m2 were placed around a further 50 test trees to collect cast needles. In October of 2007, 4 y after planting the test trees and 3 y after planting the small seedlings, all ‘‘small seedlings’’ were collected (244). At this time, they were all under or immediately beside the test tree (~2 m tall) and were typically 20–30 cm tall. In addition all other woody plants from natural regeneration within 2 m of each infected tree were collected. This included Abies balsamea (17 plants), Pinus strobes (18 plants), Picea rubens (11 plants), 34 unidentifiable (due to
99
the age of the tree) Picea species (likely mostly white spruce), P. glauca (eight plants), 20 Salix species, eleven Betula papyrifera plants, five Viburnum cassinoides plants and three Tsuga canadensis plants, as well as cast needles from the screens. These trees/shrubs were 20–30 cm tall. All samples were frozen, freeze-dried and ground to a fine powder (Sumarah et al. 2008a).
Analysis An ELISA (Enzyme-Linked ImmunoSorbent Assay) analysis was carried out to determine the amount of P. scopiformis in the needles (or leaves). Analysis was performed using a polyclonal antibody test that is specific and sensitive to P. scopiformis. The method limit of detection (LOD) for cell mass was 60 ng g 1 (i.e. 60 ppb) and the limit of quantification (LOQ), i.e. a positive, was 120 ng g 1 dry weight of needle (Sumarah et al. 2005, 2008a) Rugulosin concentration was determined by High Performance Liquid Chromatography with a Diode Array Detector. Briefly, 300 mg of freeze-dried, ground needle was extracted in ice-cold petroleum ether. The suspension was filtered and the extract discarded. The needles were then re-extracted with 10 ml of chloroform. The chloroform extract was washed with 5 % NaHCO3. The first chloroform layer was discarded while the aqueous fraction was acidified to pH 3 and extracted with a further 10 ml of chloroform. The chloroform layer was removed and dried in an amber vial under nitrogen. The dried extracts were re-dissolved in acetonitrile. Analysis was done with an 1100 series Agilent Technologies HPLC-DAD with a 250 4.6 RP column with a gradient elution. Samples were analyzed at 389 nm, the maximum UV/VIS absorption for rugulosin and peak identity was confirmed by full spectrum data from the diode array detector. The LOD and LOQ for rugulosin were both 150 ng g 1 (150 ppb; Sumarah et al. 2008a). For statistical purposes, values negative by ELISA and below the analytical detection limit were entered at half the detection limit for rugulosin (as is normal for exposure characterization). Needles that were negative for rugulosin but positive by ELISA were entered at the detection limit because there was positive evidence for the presence of the fungus in the needle.
Results and discussion Of the small seedlings planted immediately beside and currently underneath the infected trees, approximately 40 % were infected, supporting the first limited observation made at 2 y post planting (six small trees; Sumarah et al. 2008a). The arithmetic mean concentration of the positives was 1.1 mg g 1 with a geometric mean of 0.6 mg g 1 (Fig 1). This is similar to data from previous studies (Sumarah et al. 2005, 2008a; Miller et al. 2008). Biomass of the fungus was present in high concentrations in the cast needles but no toxin was detected, which was also similar to samples collected over two previous years (Sumarah et al. 2008a). P. scopiformis was not detected in non-target plants. Of the 53 spruce seedlings collected, five (9 %) were positive for Picea scopiformis (DAOM 229536, CBS 120377) or a very closely related
100
J.D. Miller et al.
0.9
Consistent with the body of evidence on the specificity of foliar endophytes of woody plants, all of the non-target woody plants were negative for the presence of the fungus under field conditions.
0.8 0.7
log ug/g
0.6
Acknowledgements
0.5
This study was supported by the Atlantic Innovation Fund, JD Irving, Limited and the National Research Council of Canada (IRAP program). HC thanks NSERC for a summer fellowship during this project. We thank Greg Slack, Luke Johnson and Madura Sornarajah for technical expertise as well as Ms. Pat MacDonald, JD Irving, Limited for collecting and identifying the non-target plants. Based on a sequence done in our lab, Dr. Christoph Gruenig (Swiss Federal Institute of Technology) provided the identification of the fungus studied with thanks.
0.4 0.3 0.2 0.1 0.0
RUGULOSIN
references Fig 1 – Rugulosin concentrations in 244 seedlings of white spruce; the length of each box shows where the central 50 % of the values fall, with the edges at the first and third quartiles.
species. Cross-reactivity to very similar strains is possible but is not material between other endophytes (Sumarah et al. 2005). Extensive tests have shown that seedlings leaving production nurseries are free of endophytes (Miller et al. 2002). The research effort that began in 1983 (Miller et al. 1985; Miller 1986; Johnson & Whitney 1989, 1992) was an attempt to understand the ecology and biodiversity of the endophyte population in Maine, New Brunswick, eastern Quebec and Nova Scotia. These and the allied secondary metabolite studies since the mid 1990’s (see Sumarah et al. 2008b) indicated that as reforestation began in 1958 there has been a gradual erosion of the genetic diversity of the foliar endophytes (Miller et al. 2008). This study, when considered with previous work on the absence of endophytes in nursery stock (Miller et al. 2002) and the experimental transmission of test strains (Sumarah et al. 2005) confirms the horizontal infection process for the foliar endophyte under study. It was known that many of the foliar endophytes in the conifers in the northern hemisphere represented a particular ecological and genetic group of mostly undescribed species (Ganley et al. 2004; Seibert 2007). There is evidence from secondary metabolite and DNA sequence information that some of these might also be aquatic hyphomycetes (Sokolski et al. 2006; Sumarah et al. 2008b). When the needles are cast (usually after 3 y in white spruce), it appears that the endophytes act as early saprobic colonizers and sporulate (Oses et al. 2008; Hyde & Soytong 2008). There is much circumstantial evidence for this in tropical plants (Promputtha et al. 2007) and even grasses (Sa´nchez Ma´rquez et al. 2008). In a plantation situation, it appears that efficient transmission is confined to naturally regenerating seedlings quite close to the infected tree. Further spread of aquatic hyphomycetes endophytes might depend on the adventitious colonization of woody debris.
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