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Systematic Botany and Mycology Laboratory, USDA, Agricultural Research Service, Beltsville, Maryland 20705-2350
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ABSTRACT |
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Forty isolates of Phomopsis were obtained from twigs and berries of highbush blueberry, Vaccinium corymbosum, and cranberry, Vaccinium macrocarpon, isolated primarily from plants grown in the eastern United States. They were characterized using conidiomatal morphology, conidial dimensions, colony appearance and growth rate, and sequences of ITS rDNA. Based on morphological and molecular similarities, most isolates grouped together with an authentic culture of Phomopsis vaccinii Shear. This taxon is described and illustrated. However, some Phomopsis isolates from Vaccinium differed in colony and conidiomatal morphology from P. vaccinii and, based on ITS sequences, were related to isolates of Phomopsis from diverse hosts. These isolates were excluded from P. vaccinii.
Key words: coelomycetes, cultural characteristics, Diaporthe, dieback, Ericaceae, fungi, ITS, pycnidia, Vaccinium corymbosum, Vaccinium macrocarpon
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Until recently, it was assumed that species of Phomopsis were restricted to specific plant hosts at the species or, at the least, genus level. Recent studies by Farr et al (1999)
, Rehner and Uecker (1994)
, and Zhang et al (1998)
suggest that delimiting species within the genus Phomopsis is more complex than had been previously recognized. Rehner and Uecker (1994)
compared the sequences of the two internally transcribed spacer (ITS) regions of the nuclear ribosomal DNA of Phomopsis isolates from eighteen vascular plant families and found little correlation between groups of Phomopsis as determined by their ITS sequences and plant host relationship or geography. In a similar study of Phomopsis isolates from soybean, Glycine max (Fabaceae), Zhang et al (1998)
were able to distinguish five taxa using ITS sequences but were unable to differentiate them based on morphological features. In identifying the cause of peach canker in the southeastern United States, Phomopsis isolates from members of the Rosaceae including peach (Prunus persica (L.) Batch.) and almond (Prunus dulcis (Mill.) D. A. Webb) were identified as Phomopsis amygdali (Del.) J. J. Tuset & T. Portilla (Farr et al 1999
). Additional isolates of Phomopsis from the same and related hosts, plum (Prunus domestica L. subsp. domestica), and Asian pear (Pyrus pyrifolia (Burm. f.) Nakai) were not conspecific with P. amygdali based on morphological, cultural, and molecular data. Using ITS sequences, P. amygdali has recently been identified from cultivated grape (Vitis vinifera L.), a non-rosaceous host (Mostert et al 2001
). Thus, while some species of Phomopsis appear to be restricted to one plant host genus or family, other species can be isolated from diverse plant hosts. Conversely, strains of Phomopsis isolated from one host species are not necessarily closely related and may represent more than one taxon.
In a study of Phomopsis on rosaceous fruit trees (Farr et al 1999
), colony characteristics, including temperature growth optima, growth rates, colony appearance, and gross morphology of pycnidia, correlated with groupings based on molecular sequences. These colony characteristics could be used to identify a taxon without sequencing. However, it was determined that the long term storage of cultures altered these characteristics; e.g., isolates obtained from culture collections were much less likely to produce pycnidia than recently isolated strains. Emphasizing the use of recently collected isolates mitigates the effects of long-term storage on pycnidium production and cultural characteristics and allows a more reliable comparison between the DNA sequences, morphology, and cultural characteristics.
Highbush blueberry, Vaccinium corymbosum L., and cranberry, V. macrocarpon Aiton, (Ericaceae) are economically important plants that produce nutritious, edible fruits. The ascomycete genus Diaporthe Nitschke, usually in the asexual pycnidial state Phomopsis, is known to cause a number of diseases on these crops, including twig blight, fruit rot, and canker of blueberry, and upright dieback and viscid rot of cranberry (Caruso and Ramsdell 1995
). For the most part the fungus causing these diseases is identified as P. vaccinii Shear, although one other species of Phomopsis, P. myrtilli Petr., has been described from Vaccinium albeit V. myrtillus. Rehner and Uecker (1994)
determined that two or more distinct taxa of Phomopsis occurred on Vaccinium, however, they did not attempt to provide names for these taxa.
In 1999, 18 isolates of Phomopsis were obtained from Vaccinium corymbosum cv. ‘Harrison’, a variety known to be susceptible to twig dieback caused by Phomopsis vaccinii Shear (Shear et al 1931
, Wilcox 1939
, Caruso and Ramsdell 1995
). For comparison, an additional 22 cultures of Phomopsis from blueberry and cranberry, primarily from the eastern United States, were obtained from plant pathologists as well as the American Type Culture Collection (ATCC) and the Centraalbureau voor Schimmelcultures (CBS). In order to determine the identity and characterize the species of Phomopsis that occur on highbush blueberry and cranberry in the eastern United States, the entire ITS region of the forty isolates was sequenced and analyzed and the resulting groupings were compared with their cultural and morphological characteristics.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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the twigs were cut into 1–2 cm pieces, placed in 95% EtOH for 30 s, followed by 66% commercial household bleach for 5 min, and then rinsed in 95% EtOH for 30 s. The twigs were placed on MYE agar (10 g malt extract, 2 g yeast extract, 20 g agar and 1 L distilled water) and incubated at 20–25 C. Phomopsis isolates were selected from the mycelium growing out from the twigs. All isolates were maintained on Difco corn meal agar slants with an alfalfa stem at 4 C and in water cultures (Burdsall and Dorworth 1994).
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Nucleic acid extraction and PCR amplification –
Mycelia for DNA extraction were grown in shaker flasks at 125 rpm for 5–10 d in 100 mL liquid CYM (Raper and Raper 1972
) at room temperature under ambient light conditions. Mycelia were harvested by vacuum filtration on Whatman No. 1 filter paper and freeze-dried prior to DNA extraction.
DNA was extracted with the DNeasy Plant Mini kit (Qiagen Inc., Chatsworth, California) according to the manufacturer's instructions using approximately 15 mg dried tissue. The internal transcribed spacer (ITS) regions 1 and 2, including the 5.8S rDNA, were amplified in 50 µL reactions on a GeneAmp 9700 thermal cycler (Applied Biosystems, Foster City, California) under the following reaction conditions: 10–15 ng of genomic DNA, 200 mM each dNTP, 2.5 units Amplitaq Gold (Applied Biosystems, Foster City, California), 25 pmoles each of primers ITS5 and ITS4 (White et al 1990
) and the supplied 10x PCR buffer with 15 mM MgCl2. The thermal cycler program was as follows: 10 min at 95 C followed by 35 cycles of 30 s at 94 C, 30 s at 55 C, and 1 min at 72 C, with a final extension period of 10 min at 72 C. After amplification, the PCR products were purified with QIAquick columns (Qiagen Inc., Chatsworth, California) according to the manufacturer's instructions. Amplified products were sequenced with the BigDye kit (Applied Biosystems, Foster City, California) on an ABI 310 or ABI 377 automated DNA sequencer.
Sequence analysis –
The resulting sequences were edited using Sequencher version 4.05 for Windows (Gene Codes Corporation, Ann Arbor, Michigan). Alignments were manually adjusted using GeneDoc 2.6.001 (Nicholas et al 1997
) and ambiguously aligned positions were excluded from the analyses. Trees were inferred using PAUP 4.0b8 (Swofford 1998
) with the following methods: the neighbor joining (NJ) method (Kimura 2-parameter distance calculation), maximum parsimony (MP) using the heuristic search (random addition with 1000 replicates) with TBR-branch swapping. In addition, maximum likelihood (ML) was used for a smaller data set consisting of Phomopsis vaccinii and closely related taxa, using empirical base frequencies A = 0.23396, C = 0.30366, G = 0.23805, and T = 0.22434), equal rates for all sites, NNI branch swapping, and a transition/transversion ratio of 2.0. The 5.8S rDNA region was excluded from both analyses, as it was not available for all taxa, and parsimony uninformative positions were excluded during the parsimony analysis of the larger data set, as searches would not run to completion when including all unambiguously aligned characters. For the smaller data set, all unambiguously aligned characters were analyzed, but the MULTREES option was turned off to allow the search to go to completion. All molecular characters were unordered and given equal weight during analysis. For parsimony analysis gaps were treated as missing data and, for neighbor joining and maximum likelihood analyses, gaps were ignored. Relative support for the branches was estimated with 1000 bootstrap replications (Felsenstein 1985
) for NJ and MP analyses. Additionally, for MP bootstrap analyses, simple sequence addition was utilized and the MULTREES option was turned off. The sequence alignments were deposited as TreeBASE S673.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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A group containing CBS 160.32, an authentic culture of P. vaccinii isolated from V. macrocarpon and deposited at CBS by Shear in 1932, seven other isolates from V. macrocarpon and six isolates from V. corymbosum was supported by NJ bootstrapping at 74% (not shown), but was not supported in MP bootstrapping (Fig. 1 ). One isolate from V. macrocarpon ATCC 56789) grouped with isolates from a variety of host genera at 72% (MP) and 82% (NJ). Two other isolates from V. macrocarpon from Washington (FAU 494) and Massachusetts (FAU 445) grouped with these isolates from Vaccinium and other hosts with poor support (51% and 53%). All of these isolates formed a larger, well-supported group, (99% and 100%) consisting of P. vaccinii, ATCC 56789, FAU 445, FAU 494, and isolates from other host genera. The seven isolates from V. corymbosum that did not group with P. vaccinii were scattered throughout the tree, grouping with isolates from Stokesia (Asteraceae), Capsicum (Solanaceae), Prunus and Pyrus (Rosaceae).
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= 7.8 µm, SD = 0.8, n = 500) x 2.1–3.9 µm wide ( = 3.0 µm, SD = 0.26), hyaline, fusiform, straight, guttulate, aseptate, forming white to yellowish cirrhi; beta conidia hyaline, filiform, straight or curved, eguttulate, aseptate. Colonies on PDA after 8 d at 25 C: white, with yellowish-gray to brownish-gray coloration around agar plug in some strains, zonate with 3 to 5 well defined zones 0.3 to 2.0 cm wide, surface mycelium felty to cottony, more dense and aerial at outer margin of each zone, margin even, lobed, or somewhat feathery, 27–48 mm diam.
Type specimen: UNITED STATES. OREGON: Clatsop, on Vaccinium macrocarpon, coll. H. F. Bain, 1924 (BPI 617410). The type specimen was examined but no useable structures remain as had been noted previously by Wehmeyer (1933)
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Authentic culture: UNITED STATES. MASSACHUSETTS: isolated from Vaccinium macrocarpon, deposited by C. L. Shear (CBS 160.32-listed as ex-type but not from type locality).
Additional cultures examined: UNITED STATES. ILLINOIS: on Vaccinium corymbosum (ATCC 56788); MASSACHUSETTS: on V. macrocarpon (FAU 446, 98020A, 98021, 98023); MICHIGAN: on V. macrocarpon (FAU 633), on V. corymbosum (ATCC 32306, FAU 590, FAU 634); NEW JERSEY: on V. macrocarpon (FAU 468, FAU 471, FAU 473–476); NORTH CAROLINA: isolated from twigs of V. corymbosum (DF 5022, DF 5023, DF 5029–5035, DF 5037–5038, DF 5042–5043), on berries of V. corymbosum (DF 5044); WISCONSIN: isolated from V. macrocarpon (ATCC 18451). Additional details about the origin of the cultures are presented in Table I .
Phomopsis vaccinii and its teleomorph, Diaporthe vaccinii Shear, were originally described from Vaccinium macrocarpon and V. oxycoccos L. by Shear et al (1931)
with a geographic range of Massachusetts, New Jersey, Oregon, Washington, and Wisconsin. Observations based on a comparison of the morphological characteristics and analysis of the ITS sequences have led us to identify the group that includes CBS 160.32 as P. vaccinii. The CBS 160.32 culture was deposited by Shear as representative of his taxon and this number is listed as the type culture in the CBS catalogue. This culture is from Massachusetts while the type specimen is from Oregon, both on V. macrocarpon. Based on the illustrations in Shear et al (1931)
and examination of the depauperate type specimen at BPI, pycnidia and conidia of P. vaccinii are similar to those produced by cultures grouping with the authentic Shear culture. This group, which includes isolates from both V. corymbosum and V. macrocarpon, is identified as Phomopsis vaccinii Shear.
Among the strains included in this study, only those recently isolated from plants in North Carolina produced pycnidia in culture when grown on alfalfa stems which, for isolates identified as P. vaccinii, included DF 5022, DF 5023, DF 5029–5035, DF 5037, DF 5038, and DF 5042–5044. Pycnidial and conidial morphology as illustrated in Figs. 3–5 confirmed the identity of these isolates as P. vaccinii. Although lacking pycnidia, the cultural characteristics (Fig. 7 ) as well as ITS sequences placed the following isolates in P. vaccinii: 93020A, 98021, 98023, ATCC 18451, ATCC 32306, ATCC 56788, FAU 446, FAU 468, FAU 471, FAU 473–476, FAU 590, FAU 633, and FAU 634. Those cultures that did not form pycnidia in culture also were somewhat variable in colony morphology, in that for some strains, the grayish color was lacking and the surface mycelium had fewer zones and tended to lack the cottony margin. In FAU 475, the surface mycelium was moist, appressed with radial fissures, having an even margin and the reverse white, yellowish white to brownish orange.
At present no teleomorph specimens or ascospore isolates are available that can be connected with P. vaccinii, thus the name often applied to this taxonomic entity, Diaporthe vaccinii Shear, is ignored. None of the twelve specimens at BPI labeled D. vaccinii included useable material of the Diaporthe sexual state. If the Diaporthe state were found on a Vaccinium host and ascospore isolates were shown to agree with P. vaccinii, the name D. vaccinii could be applied to this species.
Non-P. vaccinii isolates – Three of the isolates from Vaccinium macrocarpon, including ones from Massachusetts (FAU 445), Washington (FAU 494), and Wisconsin (ATCC 56789), differed in colony morphology from P. vaccinii. The colony of ATCC 56789 is pale yellowish with a felty to lightly cottony surface mycelium and radial fissures near the agar plug, a lobate margin, linear growth of 50 mm, and a white reverse. The colonies of FAU 494 and FAU 445 are brownish orange with a uniformly felty to cottony texture and lobate margin, linear growth of 44 mm, and a brownish orange reverse. These isolates grouped with unnamed isolates of Phomopsis from a wide range of hosts.
Seven of the isolates from Vaccinium corymbosum including one from Oregon (FAU 453) and six from North Carolina (DF 5025, DF 5026, DF 5028, DF 5036, DF 5040, and DF 5041), differed from P. vaccinii in their pycnidial, conidial, and colony morphology (Figs. 6, 8–11
). The colonies are grayish yellow to brownish orange, obscurely zonate or non zonate, with uniformly and densely cottony surface mycelium and distinctive radial strands, have an even or feathery margin, linear growth of 65–70 mm, and a golden-yellow reverse. In DF 4036 the colony varies slightly being yellowish gray or grayish orange or white, obscurely zonate or non zonate and having surface mycelium that is uniformly and densely cottony. The conidiomata are eustromatic, superficial, scattered, black, elongated, often with one or more branches, uniloculate. The conidiomatal wall is several cells thick with the outer layers brown to black and an ostiole that is single or multiple, circular in shape. Fertile cells line the inner wall of the locules. Conidiophores are short with 1 or 2 septa or multiseptate and branched. Conidiogenous cells are enteroblastic, phialidic, with moderate thickening of the channel and periclinal walls. Conidia are of two types: alpha conidia 5.5–8.7 µm long ( = 7.1, SD = 0.6, n = 190), x 1.7–2.8 µm wide ( = 2.2, SD = 0.21), hyaline, fusiform, straight, guttulate, aseptate, forming white to yellowish cirrhi; beta conidia hyaline, filiform, straight or curved, eguttulate, aseptate. Aside from being morphologically distinct from P. vaccinii, the non-Phomopsis vaccinii isolates from Vaccinium could not be adequately characterized, named or allied with known species of Phomopsis. It is possible that DF 5028, DF 5040, and DF 5041 represent an undescribed species of Phomopsis that is limited to Vaccinium as a host.
Color photographs of the cultures of isolates used in this study are available at 
http://nt.ars-grin.gov/phomopsis/
along with the results of examination of type and other specimens at BPI filed as Diaporthe vaccinii and Phomopsis vaccinii.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. Shear et al (1931)
suggested that this fungus was an important cause of storage rot in cranberry. Wilcox (1939)
determined that the Phomopsis pathogen on fruits of V. macrocarpon was the same fungus as on shoots of V. corymbosum. Using isolates from blighted twigs of V. corymbosum and from twigs and rotted fruits of V. macrocarpon, healthy plants of V. corymbosum were inoculated with cultures using spore suspensions or mycelium. Isolates produced disease symptoms on reciprocal hosts. The strains recently obtained from V. corymbosum in North Carolina were isolated from twigs having dieback symptoms as well as one strain isolated from a diseased fruit (DF 5044). Our analysis of strains of Phomopsis pathogenic on V. corymbosum represented by isolate ATCC 32396 (Weingartner and Klos 1975
) and ATCC 56788 (Chao and Glawe 1985
) grouped within P. vaccinii. One of the isolates (ATCC 18451) consistently associated with twig dieback symptoms on V. macrocarpon (Friend and Boone 1968
) also grouped within P. vaccinii. The three isolates of Phomopsis from Vaccinium macrocarpon from Massachusetts, Washington, and Wisconsin that fell outside P. vaccinii were scattered throughout a poorly supported group of isolates from a diverse range of plant hosts and geographic localities. None of these isolates produced pycnidia in culture; however, they could be distinguished from P. vaccinii based on colony characteristics. The seven isolates from Vaccinium corymbosum from Oregon and North Carolina that fell outside P. vaccinii also grouped with diverse plant hosts. Three of these isolates, specifically FAU 453, DF 5025, and DF 5026, grouped with strains identified as Diaporthe phaseolorum (Cooke & Ellis) Sacc. from Stokesia (Asteraceae) and more distantly with isolates identified as Phomopsis longicolla T. W. Hobbs on Glycine including the sequences of the ex-type culture from Ohio (U11357, U11411). The other four isolates grouped only distantly with isolates on Prunus and Pyrus from Georgia. These Vaccinium isolates do not appear to have any host plant or geographic similarities with isolates in their respective groups. However, they differ in their pycnidial, conidial, and colony morphology from P. vaccinii. The pycnidia tend to have long, unbranched or branched necks, unlike those of P. vaccinii, which are short. The conidia of P. vaccinii are longer and wider than those of the non-P. vaccinii isolates from V. corymbosum (Fig. 12 ). In addition, the linear growth of these seven isolates on PDA is faster than for P. vaccinii and can be easily differentiated from P. vaccinii.
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) and Ericaceae (Petrini 1985
). It is possible that the non-P. vaccinii isolates are endophytes and not active participants in the blueberry and cranberry diseases. But, as has been summarized by Petrini (1996)
, there are fungal endophytes that under some circumstances behave as pathogens, while under other conditions will remain innocuous.
Farr et al (1999)
noted that several of the Phomopsis cultures being investigated as the cause of peach shoot blight did not produce pycnidia in culture. In the present study none of the older FAU cultures or the ATCC and CBS cultures produced pycnidia in culture. These isolates had been maintained in culture collections from eight to 68 yr. Uecker (1989)
also found that older cultures of Diaporthe phaseolorum lost the ability to produce pycnidia. Pycnidia and cultural characteristics provide taxonomically useful information, yet Phomopsis cultures that have been in storage for years cannot be characterized using these morphological features. Thus, it is important to deposit dried cultures with pycnidia as are often produced in vitro on alfalfa stems as voucher specimens for newly isolated living cultures (Farr and Paul 2001
).
Sequence analysis of the ITS rDNA for Phomopsis isolates has been used successfully in a number of studies to identify unknown isolates from both diseased and asymptomatic hosts and can be useful in identifying isolates that no longer produce pycnidia. In this study, initial analyses including all available alignment positions separated isolates of P. vaccinii from all other taxa in the alignment, including a group of closely related strains. However, when ambiguously aligned positions were excluded, support for the molecular separation of P. vaccinii from closely related strains disappeared due to the large number of phylogenetically informative positions that were discarded among the ambiguously aligned positions. When P. vaccinii sequences were analyzed with sequences from closely related strains identified in the initial analyses, the resulting sequence alignment required far fewer positions to be discarded, resulting in high levels of bootstrap support for group identified as P. vaccinii.
Alignment of the ITS regions across the genus Phomopsis is problematic due to large numbers of insertion and deletion events, which result in dissimilar sequences that cannot be easily aligned manually. The number of ambiguously aligned positions can be quite large, and removing them from the analyses may obscure the relationships and separation of closely related taxa, requiring separate analyses of closely related taxa to accurately determine relationships. In addition, parsimony analysis of ITS sequence data for this group indicates a large amount of homoplasy across the entire genus such that large numbers of most parsimonious trees can be generated. Analysis of ITS sequence data for taxa within this genus may identify well-supported groups of closely related taxa, but phylogenetic relationships for more distantly related taxa cannot be determined with a high level of confidence. Analysis of multiple genes will be required to definitively sort out phylogenetic relationships among species of Phomopsis. It is also clear from this study that large numbers of isolates from diverse hosts should be included in analyses in order to more accurately place isolates, as host plant identity may not accurately indicate relationships within Phomopsis and the assumption that isolates from a single host genus or species are identical or even closely related may be misleading.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication November 18, 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Burdsall HH Jr,, Dorworth EB., 1994 Preserving cultures of wood-decaying Basidiomycotina using sterile distilled water in cryovials Mycologia 86:275-280
Caruso FL, Ramsdell DC., 1995 Compendium of blueberry and cranberry diseases St. Paul, Minnesota: American Phytopathological Society Press. 87 p
Chao CP, Glawe DA., 1985 Studies on the taxonomy of Diaporthe vaccinii Mycotaxon 23:371-381
Farr DF, Castlebury LA, Pardo-Schultheiss RA., 1999 Phomopsis amygdali causes peach shoot blight of cultivated peach trees in the southeastern United States Mycologia 91:1008-1015
Farr DF, Paul B., 2001 Protocols for handling living fungal cultures at SBML Inoculum 52:13
Felsenstein J., 1985 Confidence limits on phylogenies: an approach using the bootstrap Evolution 6:227-242
Friend RJ, Boone DM., 1968 Diaporthe vaccinii associated with dieback of cranberry in Wisconsin Pl Dis Reporter 52:341-344
Mostert L, Crous PW, Kang J-C, Phillips AJL., 2001 Species of Phomopsis and a Libertella sp. occurring on grapevines with specific reference to South Africa: morphological, cultural, molecular and pathological characterization Mycologia 93:146-167
Nicholas KB, Nicholas HB Jr,, Deerfield DW II., 1997 GeneDoc: analysis and visualization of genetic variation, EMBNEW News 4:14
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Raper JR, Raper CA., 1972 Genetic analyses of the life cycle of Agaricus bisporus Mycologia 64:1088-1117
Rehner SA, Uecker FA., 1994 Nuclear ribosomal internal transcribed spacer phylogeny and host diversity in the coelomycete Phomopsis Can J Bot 72:1666-1674
Sahashi N, Kubono T, Miyasawa Y, Ito S., 1999 Temporal variations in isolation frequency of endophytic fungi of Japanese beech Can J Bot 77:197-202
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Swofford DL., 1998 PAUP*4.0. Phylogenetic analysis using parsimony Sunderland, Massachusetts: Sinauer Associates
Uecker FA., 1988 A world list of Phomopsis names with notes on nomenclature, morphology, and biology Mycol Mem 13:1-323
Uecker FA., 1989 A timed sequence of development of Diaporthe phaseolorum (Diaporthaceae) from Stokesia laevis New York Bot Gard 49:38-50
Wehmeyer LE., 1933 The genus Diaporthe Nitschke and its segregates Ann Arbor, Michigan: University of Michigan Press. 349 p
Weingartner DP, Klos EI., 1975 Etiology and symptomatology of canker and dieback diseases on highbush blueberry caused by Godronia (Fusicoccum) cassandrae and Diaporthe (Phomopsis) vaccinii Phytopathology 65:105-110
White TJ, Bruns T, Lee S, Taylor J., 1990 Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics In: Innis MA, Gelfand DH, Sninsky SS, White TJ, eds. PCR protocols: a guide to methods and applications. San Diego, California: Academic Press, Inc. p 315–322
Wilcox MS., 1939 Phomopsis twig blight of blueberry Phytopathology 29:136-142
Zhang AW, Riccioni L, Pedersen WL, Kollipara KP, Hartman GL., 1998 Molecular identification and phylogenetic grouping of Diaporthe phaseolorum and Phomopsis longicolla isolates from soybean Phytopathology 88:1306-1314[Medline]
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