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Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, Box 5000, 4200 Highway 97, Summerland, British Columbia, V0H 1Z0 Canada
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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The fungus Botrytis cinerea has been widely accepted as the species responsible for causing gray mold decay of apple, although a second species causing apple decay, B. mali, was reported in 1931. Botrytis mali was validly published in 1931, nevertheless it has always been considered a doubtful species. To study the relationship of Botrytis isolates causing gray mold on apple, DNA sequence analysis was employed. Twenty-eight Botrytis isolates consisting of 10 species were sampled, including two B. mali herbarium specimens from apple originally deposited in 1932. The DNA sequence analysis of the β-tubulin and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) genes placed the isolates into groupings with defined species boundaries that generally reflected the morphologically based model for Botrytis classification. The B. cinerea isolates from apple and other host plants were placed in a single clade. The B. mali herbarium specimens however always fell well outside that clade. The DNA sequence analysis reported in this study support the initial work by Ruehle (1931)
describing the apple pathogen B. mali as a unique species.
Key words: morphology, phylogenetics, plant pathogen, systematics
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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). The genus has at times included up to 380 names (Jarvis 1980) but presently includes just over 20 species (Staats et al 2005). In a recent review of the genus, Beever and Weeds (2004) state that additional host-specific species undoubtedly remain to be described and a number of described species are poorly characterized.
Botrytis cinerea Pers.: Fr. is a common, omnivorous fungal plant pathogen. Most species of Botrytis are considered specialists possessing a narrow host range, while B. cinerea has more than 2000 hosts recorded by the United States Department of Agriculture (http://nt.arsgrin.gov/fungaldatabases/fungushost/FungusHost.cfm) and Elad et al (2004). Two species of Botrytis have been reported causing decay of apple fruit. Botrytis cinerea is considered the principal species causing gray mold fruit rot on apple (Turechek 2004). A second species, Botrytis mali Ruehle, was isolated from decay in stored fruit in Washington State 1926–1929 (Heald and Ruehle 1930, Ruehle 1931). This second species has not been reported since and has been regarded as a doubtful species (Farr et al 1989, Rosenberger 1990). Consequently if encountered this Botrytis species likely has been regarded as B. cinerea.
The phenotypic diversity exhibited by B. cinerea can be considerable (Chardonnet et al 2000, Yourman et al 2001, Kerssies and Bosker-van Zessen 1997) and must be taken into account. Transposable elements have been implicated in causing some of the morphological variability of this pathogen isolated from grape, (Levis et al 1997, Martinez et al 2003) kiwi, pea and squash (Ma and Michailides 2005). In addition such elements also have been linked to epidemiological differences in subpopulations isolated from the same host plant (Martinez et al 2005). Host specialization of B. cinerea is an additional factor implicated in the development of phenotypic variability within the species (Thompson and Latorre 1999). However morphological differences seen in Botrytis spp. isolated from a single plant also might be an indication of several different Botrytis species specialized to infect a common host, as seen with neck rot of onion (Allium cepa L.) (Presly 1985).
Molecular methods often are employed to assist in detection and identification of new species of fungi when techniques based on morphological differences are insufficient (Bridge and Arora 1998). For example universally primed PCR and RFLP analysis have been used to aid in the differentiation of the Botrytis spp., B. aclada Fresen, B. byssoidea J.C. Walker, B. squamosa J.C. Walker and B. cinerea, associated with onion neck rot (Nielson et al 2002). In other fungal genera DNA sequence analysis is an additional technique effectively used in phylogenetic, taxonomic and diagnostic studies. DNA sequence information from genes such as β-tubulin (de Jong et al 2001; Sholberg et al 2004a; Fournier et al 2005, 2006) and actin (Cox et al 1995), as well as from mitochondrial rDNA (de Jong et al 2001) and ribosomal DNA regions (de Jong et al 2001, Sholberg et al 2004b), all have revealed significant taxonomic information. Fournier and colleagues (2006) reported using DNA sequence data from four genes, including the β-tubulin and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) genes, to distinguish two Botrytis sibling species (B. cinerea Group I and II) from grape.
To study the phylogeny of 22 species of Botrytis, Staats et al (2005) used DNA sequence data from three separate genes, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60) and DNA-dependent RNA polymerase II (RPB2). Their sequence data supported the classic morphological definition of Botrytis species, and they concluded that phenotypic information combined with DNA sequence analysis is a powerful tool in the identification of Botrytis species.
The possibility of two Botrytis species, B. cinerea and B. mali, causing gray mold of apple, is not only of biological interest but could have economic implications. Both orchard and packing house management practices for the control of gray mold might need to be altered, especially if the epidemiology of two Botrytis species is different. Therefore the aim of this study was to use molecular techniques to address Botrytis species concepts in isolates from apple. Included in this study were two B. mali herbarium specimens, collected from decay in apple fruit in cold storage in Washington. These were compared with B. cinerea isolates from apple and other host plants, as well as to a number of other Botrytis spp. A preliminary report on these findings was published (O’Gorman et al 2005). DNA sequence analysis of the β-tubulin and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) genes were used to examine the relationship between Botrytis isolates capable of causing apple decay.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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). Reference isolates of 10 Botrytis species also were assembled from private, national and international collections (TABLE I). Stock cultures were stored on potato-dextrose agar slants at 2 C. The only material identified as B. mali comprises two dry cultures deposited as herbarium specimens in the U.S. National Fungus Collection (BPI) in 1932, respectively BPI 411770 and BPI 412756.
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. Attempts to revive the two 74 y old dry cultures of B. mali failed. DNA extraction therefore was conducted using what appeared to be a small piece of sclerotial material cut from the edge of the dry cultures. The Fast DNA kit was used on the herbarium samples following the manufacturer’s protocol, with one modification to the protocol consisting of one additional wash step with 300 µL of a 6 M guanidine thiocyanate solution.
The β-tubulin gene was amplified as described by de Jong et al (2001) with the primer pair Bt-Lev-Up4 and Bt-Lev-Lo1 (TABLE II). Amplification of the G3PDH gene used slightly modified versions of the primers G3PDHfor+ and G3PDHrev+ (Staats et al 2005) (TABLE II). Amplification was performed on a GeneAmp 2700 thermal cycler (Applied Biosystems, Foster City, California) with these cycle conditions for the β-tubulin gene: 95 C for 2 min followed by 35 cycles of 95 C for 20 s, 58 C for 20 s, and 72 C for 1 min, and a final extension cycle of 72 C for 7 min; conditions were similar for the G3PDH gene but used a 64 C annealing temperature for 20 s.
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) were required to amplify short overlapping fragments from both the β-tubulin and the G3PDH genes of the DNA extracted from the B. mali herbarium specimens. These primers were designed to amplify the β-tubulin or the G3PDH gene from all Botrytis spp. The cycling conditions used for this amplification reaction were similar to those used above, except for an annealing temperature of 60 C.
Sequence reaction mixtures contained 15–20 ng of purified DNA. All primers (TABLE II) were used in separate reactions with the Big Dye Terminator reaction mix (Applied BioSystems, Foster City, California) to obtain sequences for both β-tubulin and G3PDH gene fragments of the Botrytis spp. The sequencing reactions were performed as described by Sholberg et al (2005). Maximum parsimony (MP) method was used to carry out a phylogenetic reconstruction analysis of the sequence data with MEGA version 3.1 (Kumar, Tamura, Nei 2004) with these settings: heuristic search using close neighbor interchange (CNI; level = 1) a branch swapping method with initial trees generated by random addition (10 reps); complete deletion option was used to treat gaps/missing data. Bootstrap analysis (1000 reps) was carried out to ascertain the reliability of a given branch patterns of MP trees obtained.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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). The analysis placed all B. cinerea isolates from host plants (apple, peach, blackberry, strawberry and sea buckthorn) as well as B. cinerea reference sequences from GenBank (U27198
[GenBank]
, X73133
[GenBank]
and Z69263
[GenBank]
) in a single clade. Isolates of B. streptothrix (Cooke & Ellis) Sacc. and B. fabae Sardiña both formed separate branches within the B. cinerea clade. β–tubulin sequences for the two B. mali herbarium specimens however formed a single ingroup in a loosely resolved clade containing the majority of the remaining Botrytis species.
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). An exception was seen in the B. cinerea clade, and although supported by strong bootstrap values it included B. fabae (AJ705013
[GenBank]
and 2032) and B. pelargonii Røed (AJ704990
[GenBank]
) and a clear delineation between these three species was impossible. However the B. cinerea clade also contained B. calthae and B. streptothrix, both of which were placed on the end of relatively long branches supported by high bootstrap values.
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). The bootstrap values for branches delineating individual species were generally strong. The B. mali samples were placed together on their own branch, outside the strongly supported Botrytis cinerea clade, which included isolates from apple and other host plants and were again closely associated with B. fabae and B. streptothrix. Isolates of B. tulipae (Lib.) Lind, B. squamosa, B. paeoniae, B. aclada and an unidentified Botrytis sp. also were placed outside the B. cinerea clade on individual branches associated with strong bootstrap values identifying distinct species groupings.
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TAXONOMY |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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to distinguish B. mali from B. cinerea.
Typification of the name Botrytis mali Ruehle.— –
From late autumn 1926 through 1929, G.D. Ruehle studied fungal decay of Washington State apples in cold storage (Ruehle 1931). One fungus was labeled "Botrytis mali sp. nov.," and that name first appeared in a list of apple-rot fungi prepared by Heald and Ruehle (1930). But there was no description, illustration of diagnostic microscopic features, or citation of specimens. Because of the lack of a description, the name was not valid (McNeill et al 2006 [ICBN: Art. 32.1d]). The next year Ruehle (1931:1146) provided a detailed description of the fungus as it appeared on potato-dextrose agar and again cited the name as a "n. sp." thus validly publishing the name, but again no specimens were cited. Neither designation of a type nor provision of a Latin diagnosis was required in 1931. Ruehle’s photographs of the front and reverse of a mycelial colony do not show diagnostic features of the anamorph genus Botrytis, hence they cannot serve as type material for a Botrytis anamorph as qualified by ICBN Art. 59.3. The depicted sclerotia represent a separate anamorph.
There are only two specimens of B. mali (TABLE I) with a connection to Ruehle’s studies, and they were sent in 1932 by F.D. Heald at Washington State College, Pullman to BPI. Both have a mass-produced printed label that is titled "United States Department of Agriculture, Pathological and Mycological Collections." Both specimens are dried agar slant cultures. One label, for the specimen accessioned as USO411770, has the specimen data entered by typewriter: "Botrytis mali [sic] Disc. by Dr. Ruehle Culture from Orig. material sent by F.D. Heald, Pullman, Wash. Feb. 1932." The specimen box contains two test tubes, each labeled in pencil: "Botrytis mali [sic], 3-12, T, 54." The other specimen (USO412756) also has a typed label: "Botrytis mali [sic] Ruehle W.S.C. 2-5-32 54." Handwritten, in ink, below the typing is the comment, "From material sent by F.D. Heald Pullman, Wash." A small envelop labeled in pencil "17" contains the specimen and a small tag stating in pencil: "Botrytis mali [sic] Ruehle, WSC-54, 2/5/32."
Both specimens have the same number, 54, and are presumed to be replicate subcultures that were inoculated in 1932, after the name B. mali was published. Because there is no definitive link between the culture(s) Ruehle studied from 1926 through 1929 and the two BPI specimens, neither specimen qualifies as original material, and therefore neither can be selected as a lectotype (ICBN Art. 9.2). Because we were successful in sequencing the DNA from USO412756 and because it bears the morphological features of the fungus described by Ruehle, this specimen is here designated as the neotype for B. mali.
Description of the neotype.— –
The herbarium specimen (BPI USO412756) is a dried agar slant ca. 3 x 1 cm. The slant surface is partially covered by a 1.0–1.3 mm thick, dark brown, cottony mat of conidiophores and mycelium. Sclerotia imbedded in the agar, black, saucer-shaped, ca. 2 x 2 mm, exterior textura angularis (FIG. 1) (Kirk et al 2001:524), cells up to 15 µm diam, walls thickened, brown to black.
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), walls brown, 0.5–1 µm thick, smooth. Conidiogenous cells (FIG. 2) typically at the apex of short (ca. 20 µm) branches, 15 x 10 µm, mostly collapsed, walls hyaline, subhyaline or pale brown, thin to 0.5 um thick, smooth. Conidia (FIG. 2) ovoid (narrowing to the point of attachment) to broadly ellipsoid, 11–14(–15) x (7–)8–9(–10) µm (n = 20), walls smooth, subhyaline, up to 0.5 µm thick, point of attachment typically 1–1.5 µm long. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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, Jarvis 1980). Conidium and sclerotial size have been shown to be important features in distinguishing some Botrytis species (Beever and Weeds 2004). These features played a prominent role in the original identification of the apple pathogen B. mali (Ruehle 1931). Evidence presented here confirms the existence of two species of Botrytis, B. cinerea and B. mali, that are capable of producing fruit rot and consequent crop loss. Botrytis mali is apparently rare, confirmed only from one or perhaps more isolates in Washington. Although a second gathering of four B. mali isolates collected near Puyallup, Washington, in 1947 reportedly were deposited at WSU Mycological Herbarium (WSP) (F. Dugan; pers comm), these could not be located in their current collection and therefore not confirmed. B. mali possibly has been overlooked and misidentified in collecting and is more widespread than material at hand would suggest. Our limited sampling of B. cinerea from apple, only five cultures, is not sufficient to truly assess the distribution of B. mali. However the confirmation and awareness that two species of Botrytis cause rot in apple fruit will lead to a more intense study of the epidemiology, distribution and respective economic consequences of the two species.
The two 74 y old dry cultures of B. mali submitted to BPI in 1932 by Ruehle and Heald allowed the addition of B. mali genetic material in the analysis with other Botrytis species. The successful extraction of DNA from this herbarium material of B. mali and its inclusion in the analysis demonstrates the tremendous value of herbaria collections to biological studies like this one and others (Ristaino 1998, Drábková et al 2002, Wittzell 1999, Rogers and Bendich 1985).
The amplification of the herbarium sample DNA required a series of Botrytis genus specific primers to be designed to amplify short overlapping fragments of DNA. These primers were designed to eliminate bias by amplifying all Botrytis species, including any Botrytis DNA present in the herbarium sample, instead of targeting individual species. The need to amplify short DNA fragments (200–350 bp) was likely due to the low concentration and poor condition of the DNA that is recovered from archival or herbarium tissue samples (Yang et al 1998). Rogers and Bendich (1985) reported that 400 bp was the average size of DNA extracted from herbarium specimens. Drábková et al (2002) also reported using similar amplification conditions as those reported in this study when working with DNA extracted from herbarium plant material. They found that the amplification of short DNA fragments of approximately 300 bp was the most successful. In the B. mali herbarium samples the DNA was likely sheared, preventing the amplification of longer fragments. A small percentage of DNA from herbarium specimens however might be present as high molecular weight DNA (Rogers and Bendich 1985) but its low concentration may be problematic in amplifying sufficient amounts for visualization on a gel or for downstream processes such as sequencing.
Using molecular tools in conjunction with more traditional approaches to fungal taxonomy or diagnostics can be a powerful combination and can help increase the resolution of species identification (Staats et al 2005). In the past many phylogenetic, taxonomic or diagnostic studies involving fungi have relied on the analysis of ribosomal DNA, in particular the internal transcribed spacer (ITS) regions that assist in separation at the genus and species levels (Beever and Weeds 2004, White et al 1990). However, among Botrytis species, limited information can be gained from ribosomal ITS sequences (Sholberg et al 2005, Staats et al 2005). ITS data revealed few base pair differences among the species included in this study and could not resolve all individuals at the species level. Instead the ITS data lumped isolates together into indistinguishable multispecies groupings. Therefore, given that this region was not able to effectively resolve Botrytis isolates at the species level, these data were not used.
In terms of Botrytis species boundaries, the DNA sequence analysis produced in this study are in general agreement with each other and are similar to those reported by Staats et al (2005). The sequence analysis from the G3PDH and β-tubulin genes defines individual species and clearly separates B. mali from B. cinerea. Nevertheless some variation is seen among the datasets with respect to the placement of the B. mali specimens and further studies will be needed to identify the true significance. Accurate characterization of this apparently rare species will require the collection and culture of fresh material.
The datasets however always showed the two authentic B. mali specimens forming a single ingroup by themselves, outside the B. cinerea clade. The Botrytis cinerea clade consistently was formed in close associations with the species B. fabae and B. streptothrix isolates for both datasets and B. calthae in the G3PDH analysis. Botrytis mali specimens also were shown to differ from B. cinerea isolates by 44 bp changes (96.7% similarity). In comparison B. mali sequence data differed from that of B. porri and B. paeoniae isolates by a total of 22 bp (98.3% similarity) and 29 bp (97.9% similarity) respectively. The data reported in this study supports the initial work by Ruehle (1931) describing the collection and identification of B. mali as a new species.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: ogormand{at}agr.gc.ca
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LITERATURE CITED |
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