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Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, Saskatchewan, Canada S7N 0X2
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Molecular and genetic approaches were used to evaluate the genetic relatedness among isolates of the fungus Phoma macrostoma Montagne originating from Canada and Europe and to other species in the genus Phoma. Distinct differences were observed in genetic variation among nine species of the genus Phoma. Randomly amplified polymorphic DNA (RAPD) revealed the presence of intraspecific genetic variation among the isolates of P. macrostoma, with the isolates being used for biological weed control being distributed in a distinct phylogenetic cluster. Additional variation within the biocontrol isolate cluster in P. macrostoma was revealed by pulsed field gel electrophoresis (PFGE), which showed that bio-control isolates generated two different chromosomal profiles, however the profiles did not relate to their Canadian ecozone origin. Mating studies showed that biocontrol isolates of P. macrostoma from Canada did not produce sexual reproductive structures and were incapable of crossing. These studies also confirmed that no obvious differentiation exists among the bio-control isolates of P. macrostoma from Canadian Eco-zones 3 and 4.
Key words: biocontrol, genetic variation, mating, PFGE, Phoma macrostoma, RAPD
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Cook et al 1996, Hintz et al 2001, Tebeest et al 1992, Teng and Yang 1993, Templeton 1992, Wapshere 1974). One aspect of the biosafety assessment is to determine the genetic variability of the biocontrol strain in relation to other similar isolates and species that may exist in the intended area of release. For the purposes of registering a microbial pest control product in Canada, the Pest Management Regulatory Agency has designated five ecozones across the country that are based on large, ecologically distinctive areas resulting from generalized criteria involving landform, soil, water, climate, flora, fauna and human factors (FIG. 1). To avoid or minimize the risk of introducing new alleles into an area through asexual or sexual interactions, it should be known that the biocontrol strain is similar genetically to the local populations before releasing it across ecozones (Hintz et al 2001, Slatkin 1987, Taylor et al 1995, Templeton et al 1979).
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). Application of these isolates to soil resulted in chlorosis and seedling death of several broadleaf weed species but had no effect on monocotyledonous plants. The 10 isolates originated in two Canadian ecozones and their biological and morphological characteristics appeared similar, but the genetic variation occurring in them was unknown.
Randomly amplified polymorphic DNA (RAPD) is an effective molecular fingerprinting tool that identifies genetic variation at the species level and higher analyses (Arisan-Atac et al 1995, Chang et al 1996, Gosselin et al 1996, Hintz et al 2001, McDermott et al 1994, Nicholson and Rezanoor 1994, Zimand et al 1994). Pulsed field gel electrophoresis (PFGE) also has been used widely and proved to be a reliable tool in studies of genetic variation in Phoma lingam Tode ex Fr. (sexual state Leptosphaeria maculans [Desm.] Ces & de Not.) (Chen et al 1996; Howlett 1997; Koch et al 1991; Lim and Howlett 1994; Morales et al 1993; Moreno-Rico et al 2002; Plummer and Howlett 1993, 1995). More conventional studies on genetic variation within a species may be evaluated by observing mating between fungal isolates (Cozijnsen et al 2000, Mengistu et al 1995, Moreno-Rico et al 2002). These molecular tools and mating studies presumably would be useful to determine the genetic variability in the biocontrol isolates of P. macrostoma.
The objectives of the present study were to evaluate the genetic variation occurring among isolates P. macrostoma with different geographic origins and to show the genetic relatedness of P. macrostoma with other species of Phoma.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). All fungal isolates were derived from single-spore cultures and grown on V-8 juice agar at room temperature (20–24 C) under a 16 h photoperiod at 100–150 µE/m2/s.
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RAPD analyses.— –
The primers used were 10-mer oligonucleotides purchased from the Biotechnology Laboratory (University of British Columbia, Vancouver, British Columbia) (TABLE II). DNA amplification was performed in 25 µL reaction mixtures, each containing 20 ng template DNA, 1 unit AmpliTaq Gold Polymerase (5U/µL, Applied Biosystems), 1/10 volume (2.5 µL) of GeneAmp® 10xPCR Buffer II, 2.0 mM MgCl2, 0.2 µM primer, and 100 µM of each dNTPs (MBI Fermentas). The PCR program was initiated at 94 C for 10 min, followed by 45 cycles of 94 C 1 min, 35 C 1 min, 72 C 2 min, at 72 C 10 min and held at 10 C. All amplification reactions were performed in an Alpha UnitTM Block Assembly for PTC DNA EngineTM Systems (MJ Research Inc.). Amplification products were resolved by electrophoresis in 1.5% agarose gels. Each amplified DNA fragment was scored as either present or absent for each isolate. To check reproducibility of amplicons, the amplification of the DNA templates was repeated twice; there were no problems with reproducibility.
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. Electrokaryotyping was performed on a contour-clamped homogeneous electric field (CHEF) DR II system (Bio-Rad, Mississauga, Ontario) with 0.5x TBE buffer at 11 C according to Chen and Séguin-Swartz (1999), with minor modifications: The initial switch time was 600 s and final switch time was 600 s at 100 V for 72 h, followed by initial 400 s and final 400 s at 100 V for 46 h. Gels were stained in 0.1 µg/mL ethidium bromide solution and photographed. The chromosomal DNA bands were recorded for each isolate as present or absent.
Genetic mating.— –
Five biocontrol isolates of P. macrostoma from Ecozones 3 and 4 were crossed among themselves and to tester isolates of P. lingam with different mating types (Mat+ or Mat–). Although no sexual state for P. macrostoma has been reported, it was important to confirm that crossing could not occur among the biocontrol isolates. Phoma lingam was used because it has known mating types, can reproduce sexually under laboratory conditions, is an important plant pathogen of canola (Brassica napus and B. rapa) in Ecozones 3 and 4 and might have some genetic relatedness to P. macrostoma. All fungal isolates were cultured on 2% V-8 juice agar medium for 14 d. On a fresh agar plate, a 10 mm mycelial plug from each isolate in the mating pair combination was placed ca. 7.5 cm apart. The cultures were incubated at 16 h photoperiod under cool white fluorescent light (100–150 µE/m2/s) at 22 C for 3 d or until it was possible to determine the growth rate of each isolate. A sterile wooden toothpick was placed between the two isolates at the junction where they would meet and merge (Mengistu et al 1993, 1995) and incubated at 10 C with the same light conditions 4–7 wk. The toothpick and the culture media were examined at 5, 6 and 7 wk for mature pseudothecia under a binocular microscope at 40x magnification. Pseudothecia that formed on toothpicks or culture medium in the plates were extracted and crushed on a glass slide in a drop of water. The slide was examined for asci containing ascospores with a light microscope at 40–100x magnification. The presence or absence of mature pseudothecia, asci and ascospores was recorded.
Phylogenetic data analyses.— –
The presence (1) or absence (0) of DNA bands in agarose gel were converted as 0–1 matrix. The data were analyzed by the phylogenetic software package TREECON® for Windows (Version 1.3b, Van de Peer and de Wachter 1994). The evolutionary distance estimation was performed according to Nei and Li (1979). An unweighted pair group cluster method with arithmetic averages (UPGMA; Benzécri 1973) was used to infer tree topology. Bootstrap analyses were included in the distance estimation and tree topology to place confidence intervals on phylogenies (Efron and Gong 1983, Felsenstein 1985, Sworfford et al 1996).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). With primers 734 and 736, banding patterns were detected for all isolates of P. macrostoma, P. lingam and one isolate of P. chrysanthemicola. Other primers (not shown) detected P. exigua, P. medicaginis, P. nebulosa or P. pomorum, but not P. macrostoma, which was the species of prime interest. Both inter-and intraspecific genetic variation in the Phoma species and isolates was observed for all primers.
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). The isolates were somewhat variable from each other, to a limited extent within this group. For example 97-15B2 had a banding pattern slightly different than the other biocontrol isolates using Primer 734 (FIG. 2A). However the biocontrol isolates did not form distinct subgroups based on their geographic origins because the isolates from Ecozone 3 (94-44B, 85-24B, 95-268B, 89-25A2, 94-359A and 97-12B) and Ecozone 4 (95-54A1, 94-26 and 94-134) were distributed within the same cluster.
The RAPD fragment patterns from the biocontrol isolates were different when compared to the other isolates of P. macrostoma var. macrostoma, P. macrostoma var. incolorata and the other isolates of various Phoma species (FIG. 2A, B). The isolates of P. macrostoma var. macrostoma originating from Larix, Forsythia, Philadelphus and Triticum were more similar to P. dennisii from Solidago than to the biocontrol isolates from Canada thistle (FIG. 2B). Also the isolates of P. herbarum were genetically distinct from the biocontrol isolates (FIG. 2B). The primers did not detect genetic variation among P. lingam Leroy, P. nebulosa 92-74, P. pomorum 91-177, P. chrysanthemicola 90-64, P. exigua 92-180-1 and P. medicaginis 94-335A1. These latter species were more distantly related to the bio-control isolates.
Genetic variation in Phoma isolates as revealed by PFGE.— –
Twenty-seven polymorphic chromosomal DNA bands were generated with the CHEF analysis. The chromosomal profiles of P. medicaginis and P. herbarum were different from those of P. macrostoma (FIG. 3A) but exhibited some similarity to P. macrostoma. However the biocontrol isolates of P. macrostoma separated into two different categories of chromosomal profiles (Types I and II). Type I included isolates 94-44B, 85-24B, 94-26, 95-268B and 95-54A1, while the other five biocontrol isolates belonged to Type II (FIG. 3B). Similar to that revealed by RAPD analysis, isolates from Ecozone 3 (94-44B, 85-24B, 95-268B, 89-25A2, 94-359A, 97-12B and 97-15B2) and Ecozone 4 (95-54A1, 94-26 and 94-134) were distributed randomly in these two chromosome profile types. The phylogenetic tree also showed that the isolates of P. herbarum separated into two chromosome profiles (FIG. 3B).
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). Only mycelial growth and asexual sporulation from pycnidia were observed when all P. macrostoma isolates were crossed with themselves, among each other or with the different mating types of P. lingam. Pseudothecia containing ascospores were formed only by crossing different mating type isolates of P. lingam.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Concerning the release of a bioherbicide across ecozones, the biocontrol isolate should be genetically similar to local populations to reduce the potential unexpected affects, such as introducing new alleles that could alter host range or pathogen growth and survival. Various approaches have been adopted for such assessment. Recent reports on the mycoherbicide Chondrostereum purpureum (Pers. : Fr.) Pouzar by biochemical and molecular marker analyses showed no host or geographic specialization in this fungus and that the introduction of rare pathogenicity alleles from isolates used as biocontrol agents was unlikely (Shamoun and Wall 1996; Ramesfield et al 1996, 1999; Gosselin et al 1999; Hintz et al 2001). In our present study molecular and genetic tools were used to investigate the genetic variation of a potential broadleaf biocontrol fungus, P. macrostoma.
RAPD has been used widely as an effective molecular tool to evaluate genetic variance at or below the species level. In our RAPD analyses different species and isolates of the genus Phoma produced different DNA profiles on the gels denoting significant inter-and intraspecific genetic variation. The biocontrol isolates of P. macrostoma were most similar to each other but uniquely different than other isolates within the same species (P. macrostoma var. macrostoma and P. macrostoma var. incolorata) and quite different from other Phoma species, including P. lingam, P. herbarum, P. denisii var. denisii, P. medicaginis, P. nebulosa, P. pomorum, P. chrysanthemicola and P. exigua. A related study on molecular monitoring of the biocontrol isolates of P. macrostoma in plant and soil environments also supported our findings. In that study a pair of PCR primers designed from the genomic DNA of the biocontrol isolates was found to be specific to only the biocontrol isolates and no bands were generated from other common soil fungi such as Cochliobolus, Epicoccum, Fusarium, Penicillium, Pythium, Sclerotinia and Septoria (Zhou et al 2004). The intraspecific variation occurring in P. macrostoma may be due to either geographic origin (Europe vs. Canada) or a unique trait (i.e. bioherbicidal activity) that evolved with a host-pathogen interaction. Although some variation occurred within the 10 bio-control isolates, their distribution in the phylogenetic tree generated from the RAPD data were not based on geographic origin. This suggested that the P. macrostoma biological control isolates, which were isolated from Ecozone 3 or Ecozone 4, are genetically similar, so the use of those isolates across these two ecozones has little risk of introducing novel alleles into a local population. Genetic variation in fungal populations is common as illustrated by P. lingam ( Johnson and Lewis 1990, McGee and Petrie 1978, Petrie 1969, Hassan et al 1991, Kuusk et al 2002). Our RAPD study also showed that the genetic distances among three P. lingam isolates were significantly different based on the confidence intervals used for bootstrap analyses. This demonstrated that these P. lingam populations were genetically diverse and movement of these populations likely would introduce new alleles into an area. With P. macrostoma there was obvious genetic variation among isolates from different continents, although the isolates from the two Canadian ecozones were relatively similar.
PFGE also was effective in distinguishing among different species and isolates according to chromosomal profiles. The phylogenetic analysis indicated that P. macrostoma may be more closely related to P. herbarum than other Phoma spp. The present study showed two distinctly different chromosomal profiles within the species P. macrostoma. However it is interesting to note that the 10 biocontrol isolates of P. macrostoma orginating from two different ecozones were distributed into the two chromosomal subgroups. At this time no other biological traits (such as host range or growth characteristics) have been associated with these two subgroups (personal observation based on unpublished data).
Studies of mating types of fungal isolates also may be useful for subspecies classification and evaluation of genetic variation. In the case of P. macrostoma it was demonstrated that the biocontrol isolates did not produce a sexual state in mating studies among themselves or when paired with known mating types from another Phoma species possessing the capability of sexual reproduction. Other researchers also have noted that P. macrostoma has no known teleomorph (Boerema et al 2004). The use of a crossing strategy was used to demonstrate to regulators that Canadian isolates of P. macrostoma were unable to reproduce sexually thus reducing the risk of developing new alleles through recombination and to demonstrate to plant breeders that there was no exchange of genetic material with an economically important plant pathogen, P. lingam. However the crossing approach did not consider the occurrence of other mechanisms of genetic exchange such as mutation or the parasexual cycle. This would require the use of a visible marker strategy, such as deploying antibiotic resistance in one isolate and the green fluorescent protein marker in another isolate, and then subculturing the intermingled colonies to determine which clones now possessed both marker traits.
In conclusion P. macrostoma may be distinguished genetically from other Phoma spp. Ecological zones have not attributed to the genetic variation in the biocontrol isolates of P. macrostoma, as determined by molecular and genetic approaches. Therefore any P. macrostoma weed biocontrol isolates originating from Ecozone 3 and Ecozone 4 could be considered by the regulators for release across these ecozones as a biopesticide with low risk of gene flow from asexual or sexual interactions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. Phone: +1 (306) 956 7260; Fax: +1 (306) 956 7247; E-mail: BaileyK{at}agr.gc.ca
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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