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Department of Plant Pathology, University of Stellenbosch, Private Bag X1, Stellenbosch 7602, South Africa
Pedro W. Crous 1
Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands, and Department of Plant Pathology, University of Stellenbosch, Private Bag X1, Stellenbosch 7602, South Africa
Paul H. Fourie
Department of Plant Pathology, University of Stellenbosch, Private Bag X1, Stellenbosch 7602, South Africa
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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Botryosphaeriaceae are common dieback and canker pathogens of woody host plants, including stone fruit trees. In the present study the diversity of members of the Botryosphaeriaceae isolated from symptomatic wood of Prunus species (plum, peach, nectarine and apricot) was determined in stone fruit-growing areas in South Africa. Morphological and cultural characteristics as well as DNA sequence data (5.8S rDNA, ITS-1, ITS-2 and EF-1
) were used to identify known members and describe novel members of Botryosphaeriaceae. From the total number of wood samples collected (258) 67 isolates of Botryosphaeriaceae were obtained, from which eight species were identified. All species were associated with wood necrosis. Diplodia seriata (= "Botryosphaeria" obtusa) was dominant, and present on all four Prunus species sampled, followed by Neofusicoccum vitifusiforme and N. australe. First reports from Prunus spp. include N. vitifusiforme, Dothiorella viticola and Diplodia pinea. This is also the first report of D. mutila from South Africa. Two species are newly described, namely Lasiodiplodia plurivora sp. nov. from P. salicina and Diplodia africana sp. nov. from P. persica. All species, except Dothiorella viticola, caused lesions on green nectarine and/or plum shoots in a detached shoot pathogenicity assay.
Key words: Ascomycetes, Botryosphaeria, Dothidotthia, Dothiorella, Fusicoccum, ITS, Neofusicoccum, pathogenicity, Sphaeropsis, systematics, translation elongation factor EF1-
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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). Although infection commonly occurs via pruning wounds and damaged tissue (Brown and Britton 1986) these pathogens also can infect directly through lenticels (Weaver 1979) and dormant buds (Britton and Hendrix 1989) and frequently occur as endophytes in symptomless tissue (Smith et al 1996), which under stress the onset of disease is stimulated (Pusey 1989). Species of this complex represent important pathogens of fruit trees and are associated with fruit rot, leaf spot, cankers, branch dieback and even tree death (Brown and Britton 1986, Pusey et al 1995).
Taxa such as Botryosphaeria dothidea (Fusicoccum aesculi), Diplodia seriata ("B." obtusa, see Phillips et al 2007) and Lasiodiplodia theobromae ("B." rhodina, see Crous et al 2006b for clarification of Botryosphaeria-like taxa) are associated commonly with diseases of peach trees (Prunus persica) worldwide (Pusey et al 1995). Botryosphaeria dothidea is known from several Prunus spp. (Pusey et al 1986). Farr et al (1989) list these three species as well as N. ribis and Sphaeropsis peckii on Prunus spp. in USA. Slippers et al (2007) found several strains of D. seriata and Neofusicoccum australe on stone fruit trees in South Africa. Additional species described from Prunus spp. include Diplodia rosulata on P. africana in Ethiopia (Gure et al 2005) and Dothiorella sarmentorum on P. armeniaca and other Prunus spp. in Europe and/or North America (see synonyms listed by Wollenweber 1941). Wollenweber and Hochapfel (1941) regarded Diplodia roumegueri var. santonensis as synonymous with Diplodia phoradendri that was collected in 1892 on P. laurocerasus (cherry laurel) in France. Gure et al (2005) also mentioned Diplodia persicina (syn. Phoma persicina) on P. persica, Diplodia cerasorum on P. avium and Diplodia amygdali on P. dulcis and P. armeniaca, but these species have not been recollected since their original description and presently are not known from culture.
When consulting the number of Botryosphaeriaceae listed in MycoBank (www.MycoBank.org), a search confined to the names "Botryosphaeria, Diplodia and Fusicoccum" results in more than 1500 species names. In a recent compilation of the phytopathogenic fungal species occurring in South Africa, Crous et al (2000) listed approximately 20 species of "Botryosphaeriaceae" (used throughout this paper to describe this complex excl. Guignardia). This rather low number of Botryosphaeriaceae is unexpected because plant diversity in South Africa is high, with an estimated fungal diversity of more than 200 000 species (Crous et al 2006a). From these numbers it is obvious that this group has been poorly studied in this region; Slippers et al (2004a, c) have shown clearly that the approach of von Arx and Müller (1954) to reduce many species to synonymy in fact was unsubstantiated, leading to the conclusion that there should be many more potentially phytopathogenic species than known to date.
Within South Africa vineyards frequently are established near fruit tree orchards, Eucalyptus windbreaks and Proteaceae, which grow wild in the mountain fynbos. Presently 11 species of Botryosphaeriaceae are known from Vitis vinifera alone, eight of which have been reported from South Africa (van Niekerk et al 2004), while Denman et al (2003) reported five species on Proteaceae in South Africa and Slippers et al (2004b) reported five on Eucalyptus, four of them occurring in South Africa. In recent years many new plant pathogenic species of Botryosphaeriaceae have been described (e.g. de Wet et al 2003, Alves et al 2004, Slippers et al 2004b, c, Luque et al 2005, Phillips et al 2005, Burgess et al 2006, Crous et al 2006b), which suggests that if saprobic species also were to be studied this number would increase rapidly.
In an attempt to introduce the "genus for genus concept" (Seifert et al 2000) in the Botryosphaeriaceae, a study by Crous et al (2006b) split the genus Botryosphaeria into several genera based on different phylogenetic lineages, which correlated with morphology. In a further attempt to reduce the unnecessary introduction of new generic names for separate clades, only one generic name was introduced per clade, either for the anamorph or in some cases for the teleomorph state, as argued by Hawksworth (2006). This resulted in the fact that only the type species, B. dothidea, and closely related species remain in Botryosphaeria, whereas Neofusicoccum was introduced to accommodate the majority of species with fusicoccum-like anamorphs, while anamorph genera such as Diplodia (incl. Sphaeropsis), Lasiodiplodia, Pseudofusicoccum, Macrophomina, Neoscytalidium and Dothiorella were used for other prominent clades. According to Crous et al (2006b) "Botryosphaeria" or "B." is used in this paper for species that morpholically are Botryosphaeria-like but are distinct phylogenetically and need to be allocated to a different genus.
There are only three reports of Botryosphaeriaceae on stone fruits in South Africa. Doidge and Bottomley (1931) mentioned an uncommon dieback of plum in the Cape region caused by a Botryosphaeria sp., while Combrink et al (1984) found D. seriata (as "B." obtusa) as a cause of post-harvest decay of peaches. In addition Slippers et al (2007) recently isolated Neofusicoccum australe and D. seriata from dieback symptoms of stone fruit trees.
Because Botryosphaeria dieback has been well studied on grapevines in South Africa (van Niekerk et al 2004) we decided to survey the fruit trees cultivated near vineyards to determine whether these could act as potential inoculum sinks or alternate hosts for the Botryosphaeriaceae known from grapevines. During a survey in three climatically diverse areas in South Africa we isolated fungal strains from plum, peach, nectarine and apricot wood. In this study we determined the diversity of Botryosphaeriaceae on Prunus species in South Africa and describe two new species isolated from wood of Prunus persica and P. salicina.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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) supplemented with 100 mg penicillin G, 50 mg streptomycin sulphate, 10 mg chlortetracycline hydrochloride, pH 6 and incubated under cool fluorescent white light at 25 C. Additional isolates were derived from pycnidia on the bark of pruning debris. Single conidial isolates were obtained from all strains of Botryosphaeriaceae for further study. Reference strains are maintained in the culture collection of the Department of Plant Pathology, University of Stellenbosch (STE-U), Stellenbosch, South Africa, and Centraalbureau voor Schimmelcultures (CBS) Utrecht, The Netherlands. Isolates used for morphological and sequence analysis are presented (TABLE I).
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and described for isolates incubated at 25 C in the dark and under near-ultraviolet light for 7 d.
Phylogenetic analysis.— –
Genomic DNA of all isolates was isolated from fungal mycelium grown on PDA plates following the protocol of Lee and Taylor (1990). The 5.8S ribosomal gene with the two flanking internal transcribed spacers (ITS-1 and ITS-2) and the translation elongation factor 1 (EF-1) were amplified and sequenced respectively with the primer pairs ITS-1F (Gardes and Bruns 1993) + ITS-4 (White et al 1990) and EF1 728F + EF1986R (Carbone and Kohn 1999) according to the conditions and protocols explained in van Niekerk et al (2004). The sequences were added to outgroups (Cercospora penzigii STE-U 4001 and C. beticola STE-U 5073) and sequences obtained from Gen-Bank (http://www.ncbi.nlm.gov). The alignment was assembled and manually adjusted with Sequence Alignment Editor v. 2.0a11 (Rambaut 2002). Phylogenetic analyses were performed with PAUP v. 4.0b10 (Swofford 2000). The data were analyzed for each region separately as well as with a combined dataset. Ambiguously aligned data positions 102–175 (ITS) and 689 and 762 (EF-1) were excluded from the analysis. Alignment gaps were treated as missing data and all characters were unordered and of equal weight. Maximum parsimony analysis was performed with the heuristic search option with 100 random sequence additions and tree bisection and reconstruction (TBR) as the branch-swapping algorithm. The robustness of trees was evaluated by 1000 bootstrap replications with 10 random sequence additions (Hillis and Bull 1993). Tree length, consistency index (CI), retention index (RI) and rescaled consistency index (RC) were calculated for the tree. A partition homogeneity test with the same search criteria was conducted in PAUP to examine the possibility of a joint analysis of the ITS and EF-1 datasets. Sequences were lodged at GenBank (TABLE I
) and the alignment in TreeBASE (23523).
Pathogenicity tests.— – Preliminary pathogenicity tests were conducted with eight taxa on detached nectarine (cv. Alpine) and plum (cv. Ruby Nel) shoots. Depending on strain availability up to three isolates per taxon were used and treated as subsamples in statistical analysis. Vegetative shoots were collected shortly after harvest, cut into 12 cm pieces (5–8 mm diam) and surface sterilized (30 s in 70% ethanol, 2 min in 0.35% NaOCl and 30 s in 70% ethanol). Nectarine and plum cane sections were wounded through the phloem and cortex tissue with a 4 mm diam cork borer and inoculated with a colonized agar plug (4 mm diam) from 1 wk old PDA culture. Acremonium strictum (STE-U 6296) and uncolonized PDA plugs were used as negative controls. Inoculated wounds were covered with Parafilm and shoots were incubated at 25 C in moist chambers (>93% RH) for 2 wk, after which surface lesions were measured. Each treatment combination consisted of one shoot, which was replicated four times in each of three blocks (=moist chambers). Re-isolations were made from the leading edges of lesions and the resulting cultures identified. The layout of the trial was a randomized block design with all treatments randomised in three blocks (=three moist chambers). Lesion length data were subjected to analyses of variance with SAS version 8.1 (SAS Institute, Cary, North Carolina) and Student’s t-least significant difference was calculated at the 5% significance level to compare treatment means for the different taxa.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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datasets (486 characters in dataset 1, 214 in dataset 2). A selection of 19 isolates was used for the phylogenetic analysis, with further 29 sequences being added from GenBank. The dataset contained 700 characters including gaps, of which 245 were parsimony informative, six were variable and parsimony uninformative and 449 were constant. After a heuristic search 192 most parsimonious trees were retained (Length = 446 steps, CI = 0.738, RI = 0.922, RC = 0.680), of which one is shown (FIG. 1). The topology was similar for the 192 trees obtained. They differed within the D. seriata/D. pinea/D. scrobiculata, the N. vitifusiforme and the N. australe subclades and in the position of "B." tsugae, D. corticola, D. cupressi and D. rosulata within the Diplodia clade.
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TAXONOMY |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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) isolated from stone fruit wood could be assigned to eight species based on the DNA sequence data generated and morphology. Two species proved distinct from known species and are newly described below.
Diplodia africana Damm & Crous sp. nov. MycoBank MB 501323. FIGS. 2–7
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Diplodiae mutilae similis, sed conidiis longioribus, (17–) 25.5–33(–34) x (10–)12–14(–15) µm.
Conidiomata pycnidial, produced on pine needles on SNA in 2–4 wk, solitary, globose to ovoid, dark brown, up to 500 µm wide, semi-immersed to erumpent, unilocular, sometimes multilocular in vitro, with a short neck and a central ostiole; wall 6–8 cell layers thick, outer layers composed of dark-brown textura angularis, becoming thin-walled and hyaline toward the inner region. Conidiophores 1–2 celled, hyaline, 10–25 x 3.5–6 µm. Conidiogenous cells holoblastic, hyaline, cylindrical, sometimes ampulliform, proliferating percurrently near the apex, sometimes with periclinical thickening, 3–15 x 3–6 µm. Conidia aseptate, hyaline, thick-walled, smooth, subcylindrical to oblong-elliptical, sometimes slightly curved, with rounded ends, hyaline after discharge from pycnidia, a few of them becoming brown, septate and finely verruculose with age, (17–)25.5–33(–34) x (10–)12–14(–15) µm, mean ± SD = 29.2 ± 3.6 x 13 ± 1.1 µm, L/W ratio = 2.2.
Cultural characteristics. – Colonies on PDA in the dark: mycelium pale olivaceous-gray, surface pale olivaceous-gray to dark gray-olivaceous, reverse olivaceous-black, umbonate with irregular zonation and lobate edges. Under near ultraviolet: mycelium and surface greenish olivaceous to dark gray-olivaceous; reverse greenish olivaceous to olivaceous-black. Colonies 26.8 mm diam after 2 d, reaching the edge the Petri dish within 5 d; cardinal temperature requirements for growth: minimum 5 C, maximum 35 C, optimum 20 C.
Host. – Prunus persica.
Distribution. – Paarl (South Africa, Western Cape Province).
Specimens examined. – SOUTH AFRICA. WESTERN CAPE PROVINCE: Paarl, from wood section close to pruning wound of Prunus persica, 10 Jun 2004, U. Damm, CBS H-19843 HOLOTYPE, culture ex-type CBS 120835 = STE-U 5908; from insect gallery associated with pruning wound canker of Prunus persica, 10 Jun 2004, U. Damm, CBS 120835 = STE-U 5946, STE-U 6289.
Notes. –
Unlike Diplodia seriata, D. pinea, D. scrobiculata and "Sphaeropsis" pyriputrescens (Shoemaker 1964, de Wet et al 2003, Xiao and Rogers 2004), the conidia of D. africana are hyaline and thick-walled even after discharge from pycnidia and only a few conidia become brown and septate with age (FIGS. 2, 6, 7). It shares these features with D. mutila, D. corticola, D. cupressi, D. quercus, D. rosulata, D. quercina, "B." tsugae and the Diplodia anamorph of "B." quercuum.
No spore dimensions are mentioned in the original description of Sphaeria mutila (syn. D. mutila) by Fries (1823) and in the description by Montagne (1834), who considered the fungus as the type species of the new genus Diplodia. According to the description of the teleomorph Physalospora mutila by Stevens (1936) and that by Shoemaker (1964), who proposed the name Botryosphaeria stevensii, the conidia are (20–)25–27 x 10–12(–16) µm. These dimensions recently were confirmed by Alves et al (2004), who studied type material of the fungus collected by Montagne. Dimensions provided by Sutton (1980) differ considerably from those, 27–31 x 12–13.5 µm. Conidia of D. africana are similar to those of D. mutila in being hyaline, aseptate and thick-walled. However this fungus differs from D. mutila in having markedly longer conidia, (17–)25.5–33(–34) x (10–)12–14(–15) µm.
Conidia of the Diplodia anamorph of "B." quercuum, D. rosulata and D. cupressi have a different shape and L/W ratio than those of D. africana. While conidia of "B." quercuum are subglobose, having a L/W ratio of only 1.5 (Shoemaker 1964), conidia of D. rosulata are oval to ellipsoid to ovoid, with a L/W ratio of 1.93 (Gure et al 2005) and conidia of D. cupressi are ovoid with a L/W ratio of 1.76 (Alves et al 2006), conidia of D. africana are subcylindrical to oblong-elliptical and have a L/W ratio 2.2. In addition conidia of D. rosulata and D. cupressi regularly turn brown and 1-celled after discharge from the pycnidium compared to D. africana (Gure et al 2005, Alves et al 2006). Conidia of D. quercus (anamorph of "B." quercicola), measuring (24–)28.8–30.8(–38) x (11–)15.9–17.1(–21.2) (Phillips et al 2005), and D. quercina, with a mean of 29.5 x 16 µm (Jacobs and Rehner 1998), are wider than D. africana. However the D. quercina isolate studied by Jacobs and Rehner (1998) turned out to be D. corticola by sequence comparison (Alves et al 2004). Conidia of "B." tsugae (Funk 1964) are much larger than D. africana at 36–41 x 18–22 µm. Conidia of D. africana have similar dimensions as D. corticola, (27.7–)29.6–30.3(–46.1) x (9.1–)13.4–13.8(–20.5) µm (Alves et al 2004). However the mean size of the conidia of D. africana (29.2 x 13 µm) is below the lower 95% confidence limits given for D. corticola and the conidia are always less than 40 µm long, unlike those of D. corticola. In addition the conidiophores of D. corticola are reduced to cylindrical conidiogenous cells (Alves et al 2004) while 1- and 2-celled conidiophores and cylindrical as well as ampulliform conidiogenous cells (FIGS. 4, 5) were observed in D. africana. The different shape, smaller size and the absence of pores separate D. africana from Diplodia porosum, which has ovoid to broadly ellipsoid conidia, (38–)42–45(–47) x (20–)22–25(–30) µm (van Niekerk et al 2004).
Wollenweber and Hochapfel (1941) described Diplodia mutila var. major (listing several synonyms) from bark of Pyrus communis in Karlshof close to former Löwenberg in Schlesia (today Poland) with elongate-ellipsoidal, mostly hyaline, aseptate conidia that later became brown and 1(–3) septate, 26–34 x 11.5–16 (23–44 x 10–23) µm, mean = 29 x 13 µm. The description of Diplodia mutila var. major fits well with that of D. africana, except for the conidial shape that is elongate-ellipsoidal compared to D. africana that has mainly subcylindrical conidia. No type material could be located in Berlin, and thus this name could not be fully resolved. The ITS and EF-1 sequences of D. africana cluster with those of D. mutila. However they are markedly different from sequences of strain CBS 112553, which has been linked morphologically to the type material of D. mutila (Alves et al 2004) and strain STE-U 5824, confirmed morphologically as D. mutila in the present study, and of all other species of Botryosphaeriaceae available on GenBank (FIG. 1). According to its unique DNA sequence data and morphology we regard D. africana a distinct species instead of a variety of Diplodia mutila.
Lasiodiplodia plurivora Damm & Crous sp. nov. MycoBank MB 501322. FIGS. 8–13
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Lasiodiplodiae venezuelensi similis, sed conidiis latioribus, (22–)26.5–32.5(–35) x (13–)14.5–17(–18.5) µm.
Conidiomata pycnidial, produced on pine needles on SNA within 2–4 wk, solitary, globose to ovoid, dark brown, up to 400 µm wide, embedded in needle tissue, semi-immersed, unilocular, with a central ostiole; wall 4–7 cell layers thick, outer layers composed of dark brown textura angularis, becoming thin-walled and hyaline toward the inner region. Conidiophores reduced to conidiogenous cells. Conidiogenous cells holoblastic, discrete, hyaline, cylindrical, proliferating percurrently several times near the apex, 8–13 x 4–7 µm. Paraphyses hyaline, cylindrical, 2–7 celled, the 1–3 basal cells often broader than the apical cells, apical cell with rounded tip, sometimes branched, up to 130 µm long, 2–5 µm broad at the upper part and up to 10 µm broad at the lower part (basal cells). Conidia initially aseptate, thick-walled (wall <3 µm), hyaline, ellipsoidal to obovate, sometimes somewhat irregular, with granular content, becoming 1-septate after release, brown, obovate, verruculose and with longitudinal striations, brittle, with outer wall easily breaking during slide preparations, (22–)26.5–32.5(–35) x (13–)14.5–17(–18.5), mean ± SD = 29.6 ± 2.9 x 15.6 ± 1.2 µm, L/W ratio = 1.9.
Cultural characteristics. Colonies on PDA in the dark: mycelium and surface white to pale olivaceous-gray, reverse pale olivaceous-buff to pale gray-olivaceous, flat with undulate margins. Under near-ultraviolet light: mycelium and surface white to pale mouse-gray, reverse pale olivaceous-buff to smoke-gray. Colonies 76 mm diam after 2 d, reaching the edge the Petri dish after 3 d; cardinal temperature requirements for growth: minimum 10 C, maximum 
35 C, optimum 30 C.
Hosts. – Prunus salicina and Vitis vinifera.
Distribution. – Stellenbosch (South Africa, Western Cape Province).
Specimens examined. – SOUTH AFRICA. WESTERN CAPE PROVINCE: Stellenbosch, from V-shaped necrotic lesion of P. salicina, 28 May 2004, U. Damm, CBS H-19844 HOLOTYPE, culture ex-type CBS 120832 = STE-U 5803; Western Cape Province, from Vitis vinifera, STE-U 4583.
Notes. –
Septate, brown, mature conidia with longitudinal striations and the presence of paraphyses characterize this fungus as belonging to the anamorph genus Lasiodiplodia within the Botryosphaeriaceae (FIGS. 10, 12, 13). Conidia of Lasiodiplodia plurivora, (22–)26.5–32.5(–35) x (13–)14.5–17(–18.5) µm, are markedly longer and wider than L. theobromae (20–30 x 10–15 µm) and shorter and narrower than L. gonubiensis (28–)32–36(–39) x (14–)16–18.5(–21) (Griffon and Maublanc 1909, Pavlic et al 2004). They are wider than L. venezuelensis (26–33 x 12–15) but do not differ much in size from L. crassispora ([27–]30 [–33] x 14–17) and L. rubropurpurea (24–33 x 13–17) (Burgess et al 2006). However an important character of Lasiodiplodia plurivora is the paraphyses. They are septate as in L. venezuelensis and L. crassispora, but in contrast paraphyses of Lasiodiplodia plurivora are up to 130 µm long and up to 10 µm wide at the base and frequently branched, whereas paraphyses of the other two species are unbranched, <70 µm long and 
4 µm wide (Burgess et al 2006). Furthermore ITS and EF1- sequence data of L. plurivora are different from L. theobromae and from the four Lasiodiplodia species described by Pavlic et al (2004) and Burgess et al (2006) (FIG. 1).
Pathogenicity. – The majority of the 67 isolates had been obtained from wood necrosis within living branches of Prunus species, which were in cross-section either V-shaped, roundish or irregularly shaped, brown necrotic areas and sometimes situated next to cankers or resin excretion. One isolate of Lasiodiplodia plurivora and one isolate of Dothiorella viticola had been obtained respectively from internal wood necrosis and bark of small dead trees. Only nine isolates of Diplodia seriata, three isolates of D. pinea and one isolate of Neofusicoccum australe originated from pycnidia from the bark of pruning material found on the orchard floor. Each of the eight species of Botryosphaeriaceae was associated with necrosis symptoms in at least one sample, which indicated a possible pathogenic relationship.
Analyses of variance of the lesion length data on nectarine and plum cane sections indicated a significant treatment effect (P < 0.0001, ANOVA tables not shown). All species except Dothiorella viticola caused lesions on nectarine shoots, and all species except D. viticola and D. pinea caused lesions on plum shoots that were significantly longer than the controls (TABLE II). Except for D. pinea lesion lengths caused by the species were similar in plum and nectarine shoots. The fungi were re-isolated from these lesions with frequencies of 68.75–100% (TABLE II). No species of Botryosphaeriaceae were isolated from the negative controls.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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Based on literature reports B. dothidea seems to be the most important species of Botryosphaeriaceae occurring on peach in other countries (Pusey et al 1995). However, because B. dothidea, N. ribis (B. ribis) and N. parvum (B. parva) often were considered synonymous until Slippers et al (2004a) clarified the concept of B. dothidea and distinguished the three species, earlier reports could include all three species (B. dothidea sensu lato). In South Africa B. dothidea s. l. has been reported from plants such as Eucalyptus, Pistacio and Proteaceae, N. ribis from Persea, Melia, Eucalyptus, Macadamia, Malus, Citrus and Proteaceae (Crous et al 2000), and N. parvum from Vitis (van Niekerk et al 2004). In spite of these reports however we did not isolate any strains of B. dothidea, N. ribis or N. parvum from Prunus wood. In a study on stone and pome fruit trees in South Africa these species also were not found (Slippers et al 2007). In contrast D. seriata proved to be the most frequently isolated (43 of 67 isolates) and occurred on all host plants in all three areas studied. It was also the dominant species in the study by Slippers et al (2007). This species also is common on grapevines in South Africa (van Niekerk et al 2004) and Australia (Taylor et al 2005). Diplodia seriata has a worldwide distribution and a wide host range, of which 128 host records presently are known from USA alone (Farr et al 1989). Although it is common on peach worldwide (Dingley and Brien 1956, Britton and Hendrix 1982) and has been found on apricot in Wisconsin, USA, (Smith and Stanosz 2006) and on peach fruits and plum and peach wood in South Africa (Combrink et al 1984, Slippers et al 2007), this is the first report of B. obtusa on apricot and nectarine in South Africa. The fungus was shown to cause lesions on both nectarine and plum shoots. In a greenhouse experiment by Britton and Hendrix (1982) D. seriata caused gummosis canker on peach trees.
Four isolates (STE-U 5808, 5809, 5812, 5901) originating from pycnidia on bark or symptomatic tissue from a peach orchard in the Paarl area were morphologically similar to D. seriata but proved to be identical to the Diplodia pinea A morphotype (CBS 393.84) based on the ITS and EF-1 dataset and varied from the D. pinea C morphotype (CBS 109725) in only one substitution. Because of little sequence variation of D. pinea to all D. seriata isolates (two substitutions in ITS and one in EF-1) and additional substitutions within both species, there was only little bootstrap support (62%) for the grouping of the four isolates with D. pinea. But these differences in sequence were reflected in morphological differences. While the conidia size of all D. seriata isolates found on Prunus wood matched the size given for this species (22–26 x 10–12 µm, Shoemaker 1964) the conidia of these four isolates were larger, (24–)30–37(–43) x (10–)12.5–17(–21) µm, and often were paler than those of D. seriata isolates. This corresponded to the description of D. pinea conidia that are yellowish brown, eventually turning dark brown, measuring 30–45 x 10–16 µm (Punithalingam and Waterston 1970). In addition the fungi have a fluffy white to gray mycelium and sporulated less abundantly than the D. seriata isolates. Palmer et al (1987) described morphotype A as having a fluffy white to gray-green mycelium and conidia 34.3–39.4 x 12.6–12.8 µm and morphotype B with a white to black mycelium closely appressed to the agar surface and conidia 33.5–34.3 x 11.6–12.1 µm. Sequence and cultural characteristics of our isolates were different from D. pinea morphotype B, described as D. scrobiculata (de Wet et al 2003), but similar to morphotypes A and C (de Wet et al 2000), which differ in distribution, host specificity, virulence (de Wet et al 2003) and conidial size (de Wet et al 2000).
According to isozyme comparisons by Swart et al (1991), RAPD analysis by Stanosz et al (1999) and de Wet (2000) and simple sequence repeat marker analyses by Burgess et al (2001), D. pinea isolates from Pinus spp. in South Africa and neighboring countries group with the A morphotype of D. pinea. Because sequences of the Prunus isolates were identical to those of the D. pinea A morphotype we conclude that these isolates also are representative of morphotype A.
Diplodia pinea is an important pathogen on Pinus spp. worldwide (Punithalingam and Waterston 1970), including South Africa, where it has been known since 1912 (Fisher 1912), and it also has been reported on Abies, Araucaria, Chamaecyparis, Cupressus, Larix, Picea, Pseudotusga, Thuja (Coniferae, Gymnospermae) (Punithalingam and Waterston 1970, Sutton 1980). The fungus also has been reported on Eucalyptus spp. and native species of Myrtaceae in Uruguay (Bettucci et al 1999, 2004), although these reports are unconfirmed. This study is the first report of D. pinea on Prunus. While the other species were isolated mainly from necrosis inside the wood of Prunus trees, three of the four D. pinea isolates obtained in this study were derived from pycnidia on the bark of pruning debris. The fungus caused lesions on nectarine shoots that were significantly longer than the control but considerably shorter than those from most of the other species tested.
Neofusicoccum australe was reported from grapevine (van Niekerk et al 2004) and plum in South Africa (Slippers et al 2007) but considered to be infrequent and of minimal importance in stone fruits. In the present study it was found commonly on three host species (peach, plum and apricot) in different areas in Western Cape, often in V-shaped necrotic tissue. Taylor et al (2005) isolated N. australe (and D. seriata) from wedge- and half moon-shaped internal lesions in grapevine wood in Australia where the fungus frequently occurred. This is the first report of N. australe on peach and apricot. According to the long lesion lengths on nectarine and plum shoots, the fungus appears to have considerable potential as pathogen of stone fruits.
Neofusicoccum vitifusiforme was isolated from symptoms on plum and peach in two South African provinces. The fungus recently was described from grapevine in South Africa (van Niekerk et al 2004) but had not been isolated previously from Prunus.
Dothiorella viticola also is newly reported from Prunus and thus far was known from grapevines in South Africa (van Niekerk et al 2004) and Spain (Lurque et al 2005). In this study it also was isolated for the first time from necrotic wood of plum and nectarine.
The conidial dimensions of isolate STE-U 5824 closely correspond to those of D. mutila. This isolate also is identical phylogenetically to the reference strain of D. mutila (CBS 112553). Diplodia mutila was found on necrotic plum wood in Paarl. The fungus was reported on peach in New Zealand by Laundon (1973) and on apricot and peach by Sutton (1980). This is its first report from South Africa, where it was found to occur in plum wood.
Diplodia africana initially was identified as D. mutila. However by comparing sequence data and conidial measurements of the different isolates found on Prunus and those in GenBank and in the literature it became obvious that it is related closely to D. mutila, D. corticola and the Diplodia anamorph of "B." quercuum but is different from them as well as from other Diplodia species and most probably is new to science. DNA sequence data (ITS and EF-1) of D. africana differ from all known species of Botryosphaeriaceae. The fungus was found several times in one peach orchard in Paarl, Western Cape, South Africa, and was shown to be potentially pathogenic to nectarine and plum shoots.
While L. theobromae is the most common Lasiodiplodia species in tropical regions (Burgess et al 2006) and a common cause of blister canker of peach (Britton and Hendrix 1982), this species was not found on any of the Prunus species studied. However another Lasiodiplodia species was isolated from plum wood that is distinct morphologically and phylogenetically from L. theobromae and all four recently described Lasiodiplodia spp. (Pavlic et al 2004, Burgess et al 2006). Morphological differences were found in conidial size, paraphyses size, septation and shape and pycnidial color. Conidia of L. plurivora are larger than those of L. theobromae and smaller than those of L. gonubiensis. Paraphyses are septate as in L. venezuelensis and L. crassispora but much longer and frequently branched.
The ITS sequence of L. plurivora (STE-U 5803) is identical to the ITS of isolate STE-U 4583 from V. vinifera in South Africa (van Niekerk et al 2004), isolates WAC11080 (GenBank AY727846
[GenBank]
) and WAC11081 (AY727847
[GenBank]
) from Vitis vinifera in Western Australia (Taylor et al 2005) and isolate MAMB-5 (AY612337
[GenBank]
) from a stem canker on an eucalypt tree in Australia (Barbosa et al 1995). Isolate STE-U 4583 (AY343482
[GenBank]
), formerly identified as L. theobromae, was included in this study and could be confirmed to be L. plurivora based on conidial dimensions and EF-1 sequence data. Conidial dimensions of WAC11080 and WAC11081 were also the same as in L. plurivora, (27–)30–32(–34) x (15–)16–18(–20) and (26–)30–32(–34) x 16–18, respectively (Taylor et al 2005). Concerning isolate MAMB-5, electrophoretic examination of intracellular marker genes (esterases, phosphatases) and laccases (Vasconcelos et al 2001) revealed that isolate MAMB-5 was different genetically from L. theobromae. The newly described fungus L. plurivora occurs on plum, grapevine and eucalypt wood in South Africa and Australia. Due to its morphological similarity to L. theobromae and similar ITS sequence this fungus had been reported erroneously as L. theobromae and further collections are now required to resolve its distribution, host range and relative importance on woody hosts.
According to the results obtained in the pathogenicity test (TABLE II) Diplodia mutila, L. plurivora, N. australe, D. africana, D. seriata and N. vitifusiforme can be considered potentially pathogenic to Prunus salicina while D. pinea and Dothiorella viticola appear to be nonpathogenic to this host under the conditions tested here. All species, excluding Dothiorella viticola, also can be considered potentially pathogenic to P. persica. While the two Lasiodiplodia isolates from grapevine from Australia produced only small lesions on grapevine in the pathogenicity trial by Taylor et al (2005), isolate STE-U 5803 produced long lesions on plum and peach. These observations correspond to results pertaining to Lasiodiplodia isolate MAMB-5, which produces high amounts of lignocellulose-degrading enzymes (Dekker et al 2001). The amounts of extracellular laccase production of this isolate were much higher than that of B. ribis and L. theobromae (Vasconcelos et al 2001). It appears therefore that L. plurivora is well equipped for biodegradation of woody plant material, and based on our preliminary pathogenicity tests it appears to be highly virulent on nectarine and plum shoots. However further pathogenicity tests should be conducted on living plants to satisfy Koch’s postulates.
In this study several species of Botryosphaeriaceae were found on a variety of Prunus species in South Africa. Most were reported for the first time on these host plants and most of them were shown to be potentially pathogenic to Prunus species. Compared with other reports on Prunus spp. (Pusey et al 1995, Slippers et al 2007) the species composition of Botryosphaeriaceae in stone fruit orchards in South Africa seems to be different and comprises a broader variety of species. The impact of these species on the productivity of stone fruit orchards in South Africa has not been studied yet. It should be noted also that most of the species found on Prunus spp. in this study are known grapevine pathogens in South Africa and/or other regions in the world (van Niekerk et al 2004, Taylor et al 2005). Because the orchards chosen for sampling were situated next to vineyards these species could have spread from grapevine plants to stone fruit trees. On the other hand infected stone fruit trees and debris could act as an inoculum source from which grapevines could be infected and/or stone fruit trees could act as alternative hosts in the absence of grapevine plants.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: crous{at}cbs.knaw.nl
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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