Plant Pathology (2013) 62, 289–297
Doi: 10.1111/j.1365-3059.2012.02637.x
Multiple Alternaria species groups are associated with leaf blotch and fruit spot diseases of apple in Australia D. O. C. Hartevelda*, O. A. Akinsanmiab and A. Drenthab a
School of Agriculture and Food Sciences, The University of Queensland, St Lucia, Qld 4072; and bCentre of Plant Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Ecosciences Precinct, GPO 267, Brisbane, Qld 4001, Australia
Leaf blotch and fruit spot of apple caused by Alternaria species occur in apple orchards in Australia. However, there is no information on the identity of the pathogens and whether one or more Alternaria species cause both diseases in Australia. Using DNA sequencing and morphological and cultural characteristics of 51 isolates obtained from apple leaves and fruit with symptoms in Australia, Alternaria species groups associated with leaf blotch and fruit spot of apples were identified. Sequences of Alternaria allergen a1 and endopolygalacturonase gene regions revealed that multiple Alternaria species groups are associated with both diseases. Phylogenetic analysis of concatenated sequences of the two genes resulted in four clades representing A. arborescens and A. arborescens-like isolates in clade 1, A. tenuissima ⁄ A. mali isolates in clade 2, A. alternata ⁄ A. tenuissima intermediate isolates in clade 3 and A. longipes and A. longipes-like isolates in clade 4. The clades formed using sequence information were supported by colony characteristics and sporulation patterns. The source of the isolates in each clade included both the leaf blotch variant and the fruit spot variant of the disease. Alternaria arborescens-like isolates were the most prevalent (47%) and occurred in all six states of Australia, while A. alternata ⁄ A. tenuissima intermediate isolates (14%) and A. tenuissima ⁄ A. mali isolates (6%) occurred mostly in Queensland and New South Wales, respectively. Implications of multiple Alternaria species groups on apples in Australia are discussed. Keywords: Alternaria allergen a1, endopolygalacturonase, hyphomycetes, small-spored Alternaria, sporulation patterns
Introduction Alternaria leaf blotch and alternaria fruit spot are significant threats to apple production in many parts of the world (Sawamura, 1962; Filajdic & Sutton, 1991). Alternaria leaf blotch was described in the USA in 1924 (Roberts, 1924) and now occurs in most apple growing regions in the world. In Australia, the disease was first observed in the Granite Belt in Queensland in the early 1990s and has since spread to all apple growing regions (Horlock, 2006). In addition to leaf blotch, Alternaria also causes fruit spot which was first reported in Japan in the 1950s (Sawamura, 1962). In Australia, the occurrence of alternaria fruit spot is limited to the Granite Belt in Queensland and the Sydney basin and Orange districts in New South Wales. There are anecdotal reports of its occurrence in Western Australia, South Australia and Victoria (Horlock, 2006). Alternaria leaf blotch and fruit spot annually cause significant crop losses in the produc-
*E-mail:
[email protected]
Published online 29 May 2012
ª 2012 The Authors Plant Pathology ª 2012 BSPP
tion areas of Queensland and the Sydney basin and Orange districts production areas of New South Wales (Horlock, 2006), which collectively represent approximately 25% of the Australian apple industry. Alternaria leaf blotch is characterized by irregular light brown spots on leaves, bordered by a dark brown to purple margin (Persley & Horlock, 2009). The disease causes severe defoliation and up to 50% defoliation has been reported in the Sydney basin of New South Wales (Horlock, 2006). In Australia, Royal Gala, Fuji, Cripps Pink (Pink Lady) and Red Delicious are the most severely affected cultivars (Horlock, 2006). Fruit spot symptoms are characterized by small, slightly sunken, light to medium brown spots on the fruit (Persley & Horlock, 2009). Fruit losses to individual growers in Queensland and New South Wales are commonly between 15 and 25% of high value varieties such as Royal Gala, Pink Lady and Fuji (Horlock, 2006). Fruit showing visible symptoms is downgraded for juicing, resulting in significant financial losses to the grower. Worldwide, different Alternaria species have been associated with alternaria leaf blotch and fruit spot on apple. The most commonly cited causal agent of alternaria leaf blotch on apple is A. mali (Filajdic & Sutton, 1991; Bulajic et al., 1996). Other Alternaria species such
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as A. alternata sensu lato have been implicated for alternaria leaf blotch on apple (Kusaba & Tsuge, 1994). In Australia, anecdotal reports using DNA sequencing and sporulation examination of a few Alternaria isolates from alternaria leaf blotch and fruit spot indicated that the isolates were not A. mali. Preliminary identification using DNA sequencing of the ITS, actin and elongation factor genes showed that these regions were not sufficiently variable to identify the Alternaria isolates and indicated the presence of an A. alternata species complex on apple in Australia (Neilsen, 2010). Hence, the identity of the Australian Alternaria isolates within the A. alternata species complex is still unknown and the lack of understanding of the identity of the pathogen(s) involved, their disease cycle and epidemiology hinders development and deployment of effective disease management practices. Although the conidia of the genus Alternaria are distinct and easy to recognize and easily separated into large-spored and small-spored species groups, it is difficult to elucidate the various species within the genus due to a high degree of similarity in their morphological characteristics (Simmons, 1992). Small-spored Alternaria spp. are also referred to as belonging to the A. alternatacomplex causing various human and plant diseases (Anaissie et al., 1989; Thomma, 2003). Molecular techniques have been used to identify species in the A. alternata-complex (Peever et al., 2000; Pryor & Michailides, 2002; Hong et al., 2006; Laich et al., 2008). The endopolygalacturonase (endoPG) gene and two anonymous regions (Peever et al., 2004; Andrew et al., 2009) and the Alternaria allergen a1 (Alta1) gene have shown potential to delineate the closely related species within the A. alternata-complex (Hong et al., 2005). When conditions are conducive, alternaria leaf blotch and fruit spot have the potential to cause significant losses to the Australian apple industry. Of particular concern to the Australian apple growers in areas where alternaria fruit spot has not yet been reported, is whether the same Alternaria species causing alternaria leaf blotch is responsible for alternaria fruit spot. It is not known if multiple Alternaria species are involved in the pathosystem that is causing the variations in effectiveness of the same disease management strategy and efficacy of the same fungicide between apple growing regions in Australia. Therefore, the aim of the research described in this paper is to test the hypothesis that, irrespective of the growing region, the same Alternaria species causes both leaf blotch and fruit spot in Australia. In order to test this overall hypothesis, the aims of this study were: (i) to determine the identity of the Alternaria species associated with alternaria leaf blotch and fruit spot in Australian apple orchards; (ii) to examine whether the same Alternaria species is associated with both diseases; and (iii) to determine whether the same species occurs in all apple growing regions in Australia. An improved understanding of the identity of the pathogen would provide a foundation for development of improved management strategies.
Materials and methods Sources of isolates Isolates from alternaria leaf blotch and fruit spot on apples were collected in the 2003 ⁄ 04, 2004 ⁄ 05 and 2005 ⁄ 06 seasons. Over 400 isolates were obtained as described by Horlock (2006) and deposited in the Queensland Plant Pathology Herbarium Brisbane culture collection (BRIP). A total of 48 isolates were selected from the collection for this study (Table 1). Isolates were selected to represent different apple producing regions in each state and different plant parts (leaf and fruit). Two additional isolates, BRIP 25570 and BRIP 48794, obtained from the Queensland Plant Pathology Herbarium Brisbane and isolate BRIP 54639, obtained from apple with fruit spot symptoms in the 2011 season, were included in the study (Table 1). Reference strains of small-spored Alternaria species identified by E. G. Simmons, A. alternata EGS 34-016, A. arborescens EGS 39128, A. tenuissima EGS 34-015, A. mali EGS 38-029 and A. longipes EGS 30-033 were also included (Table 2). Each isolate was grown on ½-strength potato dextrose agar (PDA; Difco Laboratories Inc.) and incubated for 10–14 days at 25C in the dark. In order to obtain monoconidial isolates, 500 lL of sterile water was placed on 2-week-old colonies and the suspension containing spores and mycelial fragments was streaked on to a water agar plate (Difco Laboratories Inc.). Each water agar plate was incubated overnight in the dark at 25C and thereafter, a germinated spore was excised using a dissecting microscope under aseptic conditions and placed onto a fresh ½-strength PDA plate. If spores were absent in the source plate, a hyphal tip was cut out and transferred to the PDA plate and incubated in the dark at 25C for 7–10 days. Monoconidial isolates were stored in 15% glycerol at )80C.
Isolation of genomic DNA DNA extractions were performed with 40 mg of mycelia of each monoconidial isolate using the Promega Wizard Genomic DNA Purification Kit (Promega Corporation). The mycelia were lysed in a 2 mL safe-lock tube (Eppendorf AG) containing a 5 mm sterile stainless steel bead and 600 lL DNA Lysis using a Tissuelyser (QIAGEN) for 1 min at 30 Hz. Thereafter, genomic DNA was obtained following the procedure in the Promega Genomic DNA Purification Kit. Concentration of the genomic DNA was determined using a SmartSpec 3000 spectrophotometer (Bio-Rad Laboratories Inc.) and diluted to a concentration of 100 ng lL)1 before storing at )20C.
PCR and DNA sequencing In order to determine the identity of the Australian Alternaria species involved, two genes, endopolygalacturonase (endoPG) and Alternaria major allergen (Alta1) were selected. PCR was performed in a Bio-Rad c1000
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Table 1 Alternaria isolates used in the study with the corresponding GenBank accession numbers of gene sequences GenBank accession Isolates 25570 45965 46349 46356c 46357 46361c 46368 46373c 46384c 46390 46395c 46398 46399c 46408 46409 46414c 46452 46455c 46469 46481 46492 46493 46495 46512c 46545 46550c 46567 46571 46573 46574 46576 46577 46580 46581 46584 46590 46656 46847 46866 46872 46899 46919 47167 47168 7175 47177 47966 48081 48600c 48794 54639
a
Host organ
Location
Date collected
endoPG
Alta1
Cladeb
Fruit Leaf Fruit Fruit Fruit Leaf Leaf Leaf Leaf Leaf Leaf Fruit Fruit Fruit Fruit Leaf Fruit Leaf Leaf Fruit Fruit Leaf Leaf Leaf Leaf Fruit Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Fruit Leaf Leaf Fruit
Qld Qld NSW Qld Qld NSW NSW NSW NSW NSW NSW NSW NSW NSW NSW NSW WA WA WA WA WA WA WA WA Qld Qld Qld SA SA SA Tas. Tas. Tas. Tas. Tas. Tas. Tas. Vic. Vic. Vic. Vic. Vic. Qld Qld SA SA Qld Qld Qld Qld NSW
May-99 Dec-04 Apr-05 Apr-05 Apr-05 Apr-05 Apr-05 Apr-05 Apr-05 Apr-05 Apr-05 Apr-05 Apr-05 Apr-05 Apr-05 Apr-05 May-05 May-05 May-05 May-05 May-05 May-05 May-05 May-05 Mar-05 Apr-05 Mar-05 May-05 May-05 May-05 May-05 May-05 May-05 May-05 May-05 May-05 May-05 May-05 May-05 May-05 May-05 May-05 May-05 May-05 May-05 May-05 Dec-05 unknown Aug-06 Jan-07 May-11
JQ282179 JQ282180 JQ282181 JQ282182 JQ282183 JQ282184 JQ282185 JQ282186 JQ282187 JQ282188 JQ282189 JQ282190 JQ282191 JQ282192 JQ282193 JQ282194 JQ282195 JQ282196 JQ282197 JQ282198 JQ282199 JQ282200 JQ282201 JQ282202 JQ282203 JQ282204 JQ282205 JQ282206 JQ282207 JQ282208 JQ282209 JQ282210 JQ282211 JQ282212 JQ282213 JQ282214 JQ282215 JQ282216 JQ282217 JQ282218 JQ282219 JQ282220 JQ282221 JQ282222 JQ282223 JQ282224 JQ282225 JQ282226 JQ282227 JQ282228 JQ282229
JQ282230 JQ282231 JQ282232 JQ282233 JQ282234 JQ282235 JQ282236 JQ282237 JQ282238 JQ282239 JQ282240 JQ282241 JQ282242 JQ282243 JQ282244 JQ282245 JQ282246 JQ282247 JQ282248 JQ282249 JQ282250 JQ282251 JQ282252 JQ282253 JQ282254 JQ282255 JQ282256 JQ282257 JQ282258 JQ282259 JQ282260 JQ282261 JQ282262 JQ282263 JQ282264 JQ282265 JQ282266 JQ282267 JQ282268 JQ282269 JQ282270 JQ282271 JQ282272 JQ282273 JQ282274 JQ282275 JQ282276 JQ282277 JQ282278 JQ282279 JQ282280
3 3 1 4 3 2 1 1 1 3 2 3 3 3 3 2 1 4 1 1 1 1 1 1 3 3 1 1 2 2 1 1 1 1 1 3 2 1 1 1 4 1 3 3 1 1 4 2 1 1 2
NSW: New South Wales; Qld: Queensland; WA: Western Australia; Vic.: Victoria; SA: South Australia; Tas.: Tasmania. a Numbers represent the BRIP codes of the isolates as coded by the Queensland Plant Pathology Herbarium, Brisbane, Australia. b Phylogenetic clade the isolates were appointed to using combined DNA sequence data of Alta1 and endoPG. c Isolates used for cultural and morphological examination in this study.
Thermal Cycler (Bio-Rad Laboratories Inc.). PCR reactions consisted of 50 ng genomic DNA, 1 · PCR reaction buffer, 1Æ5 mM MgCl2, 0Æ6 lM of each primer, 0Æ1 mM Plant Pathology (2013) 62, 289–297
dNTP and 0Æ66 U Taq DNA polymerase (Fisher Biotech) in a 30 lL volume for both endoPG and Alta1 reactions. PCR conditions for the endoPG gene consisted of initial
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Table 2 Alternaria species reference strains used in this study GenBank accession Isolates EGS EGS EGS EGS EGS
a
34-016 39-128 34-015 38-029 30-033
CBS codes CBS CBS CBS CBS CBS
b
916.96 102605 918.96 106.24 540.94
Host
Location
endoPG
Alta1
Alternaria species
Arachus hypogaea Lycopersicon esculentum Dianthus caryophyllus Malus domestica Nicotiana tabacum
India California United Kingdom United States North Carolina
AY295024 AY295028 n ⁄ ac AY295020 n ⁄ ac
AY563301 AY563303 AY563302 n ⁄ ac AY563304
A. A. A. A. A.
alternata arborescens tenuissima mali longipes
a
Representative Alternaria species as identified by E. G. Simmons, Mycological Services, Crawfordsville, IN 47933, USA. Codes of the isolates as coded by the Centraal Bureau of Schimmelculturen, Utrecht, The Netherlands. c Sequences of the isolates were obtained from the Centraal Bureau of Schimmelculturen, Utrecht, The Netherlands. b
denaturation of 95C for 60 s, followed by 35 cycles at 95C for 30 s, 55C for 30 s, 72C for 30 s and a final extension at 72C for 7 min using PG3 forward primer 5¢-TACCATGGTTCTTTCCGA-3¢ and PG2b reverse primer 5¢-GAGAATTCRCARTCRTCYTGRTT3¢ (Peever et al., 2004; Andrew et al., 2009). PCR cycling conditions for Alta1 were performed as described by Hong et al. (2005) with Alt-for forward primer 5¢-ATGCAGTTCACCACCATCGC-3¢ and Alt-rev reverse primer 5¢-ACGAGGGTGAYGTAGGCGTC-3¢. The PCR fragments were separated in 1% agarose (Bioline) gels using 0Æ5 · Tris–borate–EDTA (TBE) buffer (Bioline) containing 0Æ08 lL mL)1 GelRed DNA stain (Biotium) and electrophoresis was performed at 95 V for 45 min. DNA fragments were visualized with UV illumination using a Molecular Imager Gel Doc (Bio-Rad Laboratories Inc.). Fragment sizes were determined using a Generuler 100 bp ladder. Amplicons were cleaned using Roche High Pure PCR Product Purification Kit (Roche) according to the manufacturer’s instructions and the purified DNA was eluted using 60 lL elution buffer and sequenced at the Australian Genome Research Facility (Brisbane, Australia) with the same primers as used for amplification. MEGA v. 5 (Tamura et al., 2011) software was used to manually assemble the forward and reverse sequences into consensus fragments. In order to provide consistency and quality of the sequences, the chromatograms of the sequences were checked, aligned and primer sequences were trimmed off at both ends of the sequences. In order to determine nucleotide identity, BLAST searches (Altschul et al., 1990) of the National Centre for Biotechnology Information (NCBI) were performed for each set of nucleotide sequences. All sequences obtained in the study have been submitted to GenBank (Table 1).
Phylogenetic analysis and relations of the Australian isolates In order to examine the phylogenetic relationships of the Australian Alternaria isolates, multiple nucleotide sequence alignments were performed using MUSCLE (Edgar, 2004). Sequences of the representative Alternaria species were obtained from GenBank and the Centraal
Bureau voor Schimmelculturen, Utrecht, The Netherlands (Table 2). With the concatenated sequences of the Alta1 and endoPG genes of the Australian isolates and the reference strains, a maximum likelihood phylogeny was obtained by using the best-fit procedure in MEGA 5. This option tests the nucleotide sequences for goodness of fit to 21 models of evolution and returns the estimated values of all parameters for each model. From the models tested, Kimura-2 parameter with a discrete gamma distribution (K2 + G) provided the best-fit model with the lowest Bayesian Information Criterion (BIC) score for the data. Tree stability was tested by using 1000 bootstrap replications.
Assessment of morphological and cultural characteristics of Australian isolates In order to examine and compare the morphological and cultural characteristics of the isolates in the phylogenetic clades, colony characteristics and sporulation patterns of 2–4 isolates of each clade were examined. The growth conditions and the morphological and cultural examinations were as described by Simmons (2007), except that the isolates were grown on low strength PDA instead of potato carrot agar and V8 medium as described by Hong et al. (Pryor & Michailides, 2002; Hong et al., 2006). Low strength PDA improves consistency of the media used to examine the morphological structures (conidiophores, sporulation patterns and spores) in small-spored Alternaria species. The isolates were grown on ¼ PDA in triplicate and incubated with an 8 h ⁄ 16 h light ⁄ dark cycle at 25C (Simmons, 2007). Colonies were examined for their size after 7 and 14 days of incubation for their colour, texture and concentric ring development. Sporulation patterns for each isolate were examined using a drop of spore suspension, placed on a water agar plate containing 12Æ5 g agar L)1 and spread over the surface of the agar. In addition, the sporulation pattern of each isolate was examined on V8-juice agar and ¼ PDA culture plates. Sporulation patterns were examined 9 days after incubation by placing a cover slip on top of the colony and placing the plate under a Leica DM5500B compound microscope (Leica Microsystems). Sporulation patterns were compared with the description of representative species as set out in the Alternaria Identification Plant Pathology (2013) 62, 289–297
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Manual (Simmons, 2007) and using the key for smallspored Alternaria species (Pryor & Michailides, 2002).
Results Identity of the Australian Alternaria isolates PCR amplifications of the Alta1 gene produced fragments of 472 bp that contained an intron of 60 bp. Introns in Alternaria and related taxa can show high variability (Hong et al., 2005). Therefore, in order to achieve comparable unambiguous alignment with published sequences of Alta1 from which introns had been removed, the intron was removed from the Australian Alternaria sequences before further analysis. Amplification of the endoPG gene produced 448 bp fragments. In order to test each gene for its ability to identify small-spored Alternaria species, the Alta1 and endoPG sequences of the representative Alternaria strains: A. alternata (EGS 34-016), A. tenuissima (EGS 34-015), A. arborescens (EGS 39-128), A. mali (EGS 38-029) and A. longipes (EGS 33-033) were compared, and individual BLAST searches were performed. With Alta1, the reference strains of A. alternata and A. tenuissima could not be differentiated. The reference strain of A. mali was differentiated from the A. alternata ⁄ A. tenuissima sequences by only 1 bp. The Alta1 sequences of the reference strains of A. arborescens and A. longipes were different and were differentiated from the other reference strains, whilst with endoPG the sequences of reference strains of A. arborescens, A. alternata and A. tenuissima could be differentiated. The endoPG sequence of A. mali was the same as A. tenuissima. The endoPG sequence of A. longipes was different, but could not be differentiated from A. alternata and A. tenuissima using BLAST searches. In order to relate the Australian Alternaria isolates to closely related Alternaria species, BLAST searches were performed for each gene. Sequences of both genes showed that the same 26 (51%) of the Australian isolates were closely related to A. arborescens. For the remaining 25 Australian isolates, the Alta1 gene showed two isolates to be similar to A. mali, four isolates (8%) as similar to A. longipes EGS 33-033 (GenBank accession AY563304) and 19 isolates (37%) as A. alternata ⁄ A. tenuissima-like isolates. The endoPG gene showed 15 isolates (29%) to be similar to A. alternata and 10 isolates (20%) related to A. tenuissima ⁄ A. mali. The consensus sequence alignment of the Alta1 and endoPG combined showed that out of 51 isolates, 26 (51%) were closely related to A. arborescens, 13 (26%) were similar to A. alternata ⁄ A. tenuissima, 8 (16%) were similar to A. tenuissima ⁄ A. mali and 4 (8%) were related to A. longipes.
Phylogenetic analysis and relations of the Australian Alternaria isolates The analysis of the concatenated sequences of the Alta1 and endoPG genes resulted in a maximum likelihood Plant Pathology (2013) 62, 289–297
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phylogenetic tree showing four distinct clades (Fig. 1). Clade 1 represented A. arborescens and A. arborescenslike isolates, clade 2 A. tenuissima ⁄ A. mali isolates, clade 3 A. alternata ⁄ A. tenuissima intermediate isolates and clade 4 A. longipes and A. longipes-like isolates (Fig. 1; Table 1).
Morphological comparisons of the Australian Alternaria isolates The morphological and cultural characteristics of the isolates selected to represent each of the four phylogenetic clades (Table 1) were consistent for all the triplicate plates. The clade 1 isolates (BRIP 46512, BRIP 48600, BRIP 46373 and BRIP 46384) produced colonies on culture media plates that were irregular in shape, dark green to black, felty and with a 5–10 mm white margin. The colonies formed visibly separated 6–8 concentric rings and a colony diameter of 40–70 mm. Sporulation patterns showed long conidiophores with extensive terminal branching and the primary conidial chains contained 2–7 conidia in length and the secondary and tertiary branches contained 1–6 conidia. Clade 2 isolates (BRIP 46414, BRIP 46361 and BRIP 46395) produced grey-green to brown-greenish colonies with woolly whitish mycelia on top of the colony. The cultures contained 3–5 vague concentric rings and were 42–60 mm in diameter. Sporulation patterns of clade 2 isolates BRIP 46414 and BRIP 46361 were similar; the primary chains contained 4–11 conidia, which commonly had 1–3 secondary chains of 1–4 conidia in length. In contrast, the sporulation pattern of isolate BRIP 46395 showed long primary chains which had 6–13 conidia and occasional secondary chains of 1–7 conidia. Colonies of clade 3 isolates BRIP 46399 and BRIP 46550 were olive green to dark green, felty and a bit woolly with a 5 mm margin. The colonies had 4–7 concentric rings and were 57–66 mm in diameter. The sporulation patterns of the two clade 3 isolates were different. Isolate BRIP 46399 had primary chains of 5–9 conidia and extensive branching into 1–3 secondary chains of 1– 4 conidia in length, whilst isolate BRIP 46550 had long primary chains of 5–13 conidia in length with few occasional secondary branches of 1–4 conidia in length. Colonies of clade 4 isolates BRIP 46356 and BRIP 46455 were grey-green colonies with a light woolly texture, five concentric rings and 65–70 mm in diameter. The sporulation showed long primary chains of 5–15 conidia and no secondary branching.
Distribution of the Australian Alternaria isolates on apples Alternaria arborescens-like isolates were the most dominant Alternaria isolates found in all the states of Australia and 22 of 37 (59%) isolates were obtained from leaf symptoms. In contrast, 9 of the 14 (64%) isolates obtained from fruit were identified as A. alternata ⁄ A. tenuissima intermediate and A. tenuissima ⁄ A. mali isolates, found mainly in New South Wales and Queensland
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46512 WA L 47175 SA L 46495 WA L 46493 WA L 46469 WA L 46481 WA F 48600 QLD L 46581 TAS L 46580 TAS L Alternaria arborescens EGS 39-128 46492 WA F 53 46571 SA L
Clade 1 A. arborescens and
46567 QLD L 46576 TAS L
A. arborescens-like isolates
71 48794 QLD L
94
46577 TAS L
52
46384 NSW L 46847 VIC L 47177 SA L 46919 VIC L 46452 WA F 46866 VIC L
46872 VIC L 46349 NSW F 46368 NSW L 46373 NSW L 46584 TAS L 46361 NSW L 46573 SA L 54639 NSW F Alternaria tenuissima EGS 34-015 Alternaria mali EGS 38-029 83
46656 TAS L
Clade 2 A. tenuissima/A. mali isolates
46395 NSW L 48081 QLD F 46414 NSW L 66 46574 SA L 99
Alternaria alternata EGS 34-016 46399 NSW F 46398 NSW F 46408 NSW F 46409 NSW F 46390 NSW L
Clade 3
25570 QLD F 46357 QLD F
A. alternata/A. tenuissima
47167 QLD L
intermediate isolates
47168 QLD L 45965 QLD L 46545 QLD L 46550 QLD F 46590 TAS L 46356 QLD F
Clade 4
47966 QLD L Alternaria longipes EGS 30-033 96 0.005
46455 WA L
A. longipes and
46899 VIC L
A.longipes-like isolates
Figure 1 Maximum likelihood phylogenetic tree using the Kimura-2 parameter model with discrete gamma distribution of the concatenated sequences of the endoPG and Alta1 genes of 51 Australian Alternaria isolates from apple and Alternaria species reference strains (EGS). Bootstrap values (only >50 are shown) at the nodes are based on 1000 replicates. Australian isolates were obtained from leaf (L) and fruit (F) in New South Wales (NSW), Queensland (QLD), Western Australia (WA), South Australia (SA), Tasmania (TAS) and Victoria (VIC). Clade 1 consists of A. arborescens and A. arborescens-like isolates, clade 2 A. tenuissima ⁄ A. mali isolates, clade 3 A. alternata ⁄ A. tenuissima intermediate isolates and clade 4 A. longipes and A. longipes-like isolates.
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Table 3 Geographical distribution of 51 Alternaria isolates obtained from alternaria leaf blotch and fruit spot symptoms on apple in Australia
Australian state
A. arborescens-like isolatesa
A. tenuissima ⁄ A. mali isolates
A. alternata ⁄ A. tenuissima intermediate isolates
A. longipes-like isolates
Total isolates
Queensland New South Wales Western Australia Tasmania Victoria South Australia Total
3 4 (1) 7 (3) 5 4 3 26 (4)
1 (1) 4 (1) 0 1 0 2 8 (2)
7 (3) 5 (4) 0 1 0 0 13 (7)
2 (1) 0 1 0 1 0 4 (1)
13 (5) 13 (6) 8 (3) 7 5 5 51 (14)
a
The number of isolates obtained from fruit is presented in parentheses.
(Table 3). Alternaria arborescens-like isolates constituted only 23% of the Queensland and 31% of the New South Wales isolates, compared to over 60% of the isolates from the other states in Australia (Table 3). Alternaria alternata ⁄ A. tenuissima intermediate isolates constituted the majority (7 out of 13) of the isolates obtained from Queensland (54%) and were associated with both leaf blotch and fruit spot (Table 3). The two isolates most closely related to A. mali were obtained from leaf blotch symptoms from South Australia and New South Wales.
Discussion Using molecular and morphological tools, this study provides evidence that multiple small-spored Alternaria species groups are associated with both alternaria leaf blotch and alternaria fruit spot disease on apple in Australia. The 51 Australian Alternaria isolates were grouped into four well-supported phylogenetic clades by using concatenated sequences of the endoPG and Alta1 genes of the isolates. The phylogenetic clades represent A. arborescens and A. arborescens-like isolates in clade 1, A. tenuissima ⁄ A. mali isolates in clade 2, A. alternata ⁄ A. tenuissima intermediate isolates in clade 3 and A. longipes and A. longipes-like isolates in clade 4. These clades were mostly supported by morphological and colony characteristics of the isolates in the clades. This is the first report of the involvement of multiple Alternaria species groups in leaf blotch and fruit spot of apple pathosystems in the world. The Australian Alternaria isolates of each clade were associated with both leaf blotch and fruit spot and present in each region, indicating potential of causing both diseases in all the Australian apple production areas. Alternaria mali has been identified as the causal agent of alternaria leaf blotch and fruit spot in Japan (Sawamura, 1962), China (Wang et al., 1997) and the United States (Filajdic & Sutton, 1991). The A. mali reference strain used in this study grouped with the isolates similar to A. tenuissima in clade 2 (Fig. 1). Morphologically, A. mali has previously been placed into the A. tenuissima species group (Andersen et al., 2006). There is still confusion in the naming of this species, as different species have been called A. mali from different locations (Simmons, 1999). Whether the species appointed as A. tenuissima Plant Pathology (2013) 62, 289–297
species group (Simmons, 1995) are A. mali or A. tenuissima or another species within the A. tenuissima species group is unclear and detailed taxonomical studies on a global scale using specimens from many different host plants are required to clarify the identity of these species. Identification of the Australian isolates using DNA sequence information of the Alta1 gene shows the potential to partly differentiate the A. alternata-complex. The Alta1 sequences of the Australian isolates resulted in a group of A. arborescens-like isolates, isolates with similar sequences to A. alternata ⁄ A. tenuissima reference strains (these strains could not be differentiated) and isolates similar to A. longipes. This indicates that this gene does not provide sufficient variability to distinguish the A. alternata-complex completely, whereas the endoPG gene separated the isolates into A. arborescens-like, A. tenuissima-like and A. alternata-like species groups. Isolates BRIP 46356, BRIP 47966, BRIP 46455 and BRIP 46899 were similar to A. longipes using the Alta1 gene but were grouped into the A. tenuissima ⁄ A. alternata species groups by endoPG. The concatenated sequences of the two genes resulted in separation of isolates into A. tenuissima ⁄ A. mali (clade 2) and A. alternata ⁄ A. tenuissima intermediate (clade 3) clades. However, the reference strain A. alternata EGS 34-016 does not fall into the clade with isolates most closely related to A. alternata, but is indicated as a separate branch of the tree. This demonstrates variation within the A. alternata species group and the close relationship with the other species groups detected in this study. Overall, these findings indicate that sequencing using Alta1 and ⁄ or endoPG is not completely sufficient to separate the A. alternata ⁄ A. tenuissima species group clusters. This is similar to Andrew et al. (2009) who suggested that to distinguish A. alternata and A. tenuissima species groups, sequence information of more variable regions are still needed. Other studies have shown close association of A. longipes with A. gaisen, A. tangelonis and A. alternata (Roberts et al., 2000; Andersen et al., 2001; Andrew et al., 2009). Morphological analyses showed that A. longipes belongs to the A. tenuissima species group (Roberts et al., 2000). A more detailed and global approach using sequence data from type cultures of all available small-spored Alternaria species obtained from a range of different host plants may provide clarity and
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confirmation of the identity of small-spored Alternaria species. Morphological descriptions of the species in each clade gives credence to the phylogenetic separation of the species into the clades. The description of the isolates in clade 1 is congruent with findings in other studies (Andersen et al., 2002; Vergnes et al., 2006). The long conidiophores and extensive terminal branching confirms the identity of the isolates as belonging to the A. arborescens species group (Pryor & Michailides, 2002). The similarity of the morphological characters and sporulation patterns of isolates within clade 4 confirmed distinctness of the clade and confirmed it to be closely related to A. longipes with long unbranched conidial chains (Andersen et al., 2001; Simmons, 2007). Morphological and cultural characters of the isolates representing clade 2 and 3 also support the separation of the isolates into different clades. However, the sporulation patterns of clade 2 isolate BRIP 46395 were similar to clade 3 isolate BRIP 46550, which may indicate a close association between the two clades. Although different sporulation patterns can indicate different species within the A. tenuissima and A. alternata species groups, identification of smallspored Alternaria species using only morphological characters may be misleading. Alternaria arborescens-like isolates are the most prevalent in Australia and are mostly associated with leaf blotch symptoms. This may explain the reason for the prevalence of leaf blotch of apples in all apple-growing states of Australia. In contrast, most (64%) of the isolates obtained from fruit were identified as A. alternata ⁄ A. tenuissima intermediate and A. tenuissima ⁄ A. mali. Alternaria alternata ⁄ A. tenuissima intermediate isolates predominate in New South Wales and Queensland rather than A. arborescens-like isolates, thus providing a rationale for the frequent occurrence of alternaria fruit spot in these states in Australia. In addition, A. alternata ⁄ A. tenuissima intermediate isolates constituted the majority (54%) of the isolates obtained from Queensland, which could explain why both diseases are more severe and frequently occur under favourable conditions in Queensland than any other states in Australia. In conclusion, the results show that the Alternaria species groups in Australia were not specific to alternaria leaf blotch or alternaria fruit spot or a geographical region. Alternaria arborescens-like isolates were found in all states and may be responsible for the majority of disease problems in Western Australia, Tasmania, Victoria and South Australia, where mainly the leaf blotch variant occurs. Isolates obtained from New South Wales and Queensland were mostly identified as A. alternata ⁄ A. tenuissima intermediate or A. tenuissima ⁄ A. mali, which indicates that these species groups may be responsible for the majority of the fruit spot disease problems in those states. Hence, this may account for the variation in efficacy of different fungicides used to control the diseases in different apple growing regions in Australia. More detailed studies to verify the reason for this variability are currently underway. The findings provide an understand-
ing concerning the identity and distribution of the Alternaria species groups that are associated with alternaria leaf blotch and fruit spot on apple in Australia, which may serve as an impetus for further studies on pathogenicity, aggressiveness and virulence of the species groups for both diseases in this country. The concern to appleproducing regions in Western Australia, South Australia and Victoria, where fruit spot has not been reported as an important disease, is that similar species causing leaf blotch and fruit spot in Queensland and New South Wales occur in these regions. Hence, under favourable conditions, A. arborescens-like isolates may cause fruit infection. These findings significantly increase the understanding of Australia’s situation regarding these diseases and will be used in future work towards the development of improved control options.
Acknowledgements This work was funded by Horticulture Australia Limited (Project AP06007) with levies from Apple & Pear Australia Ltd. The authors would like to thank C. Horlock for providing the Australian isolates to the Queensland Plant Pathology Herbarium Brisbane, and J. H. C. Woudenberg and Professor P. W. Crous of the Centraal Bureau voor Schimmelculturen, The Netherlands for providing sequences of reference strains.
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