Plant Pathology (2006) 55, 485–493 Doi: 10.1111/j.1365-3059.2006.01391.x Identification of Alternaria spp. on wheat by pathogenicity assays and sequencing Blackwell Publishing Ltd D. Mercado Vergnesa*, M.-E. Renarda, E. Duveiller b and H. Maraitea a Unité de Phytopathologie, Université Catholique de Louvain, Croix du Sud 2 bte 3, B-1348 Louvain-la-Neuve, Belgium; and CIMMYT, South Asia Office, Singha Durbar Plaza Marg, PO Box 5186, Kathmandu, Nepal b The pathogenicity of Alternaria spp. isolated from wheat leaves collected in regions where alternaria leaf blight has been reported was compared with that of IMI reference isolates of A. triticina and A. alternata using two durum and two bread wheat genotypes. To identify isolates putatively corresponding to A. triticina, morphological and DNA sequence analyses based on ribosomal DNA from the internal transcribed spacer (ITS) region (ITS1, 5·8S rRNA gene, ITS2) and toxicity bioassays of culture filtrate were combined. Glasshouse inoculations provided reliable information to assess the pathogenicity of A. triticina isolates on wheat. Alternaria leaf blight symptoms were produced by the A. triticina isolates only on durum wheat cv. Bansi, while A. alternata, A. tenuissima and A. arborescens isolates were found to be nonpathogenic on the wheat cultivars tested. Alternaria triticina isolates were distinguished from other Alternaria species by Simmons and Roberts’ sporulation pattern 6 and two to three conidia per sporulation unit associated with primary conidia bearing long (> 7 µm) apical secondary conidiophores. Phylogenetic analysis also proved effective at discriminating wheatpathogenic A. triticina from other nonpathogenic Alternaria species. Alternaria triticina isolates yielded longer ITS sequences than A. alternata, A. tenuissima and A. arborescens isolates, leading to clear-cut differences as visualized with agarose gel electrophoresis. Additionally, only culture filtrates of A. triticina isolates caused nonspecific necrotic lesions on leaves of 3-week-old wheat plants. Keywords: alternaria leaf blight, foliar blight, nonhost-specific toxin, Triticum aestivum, Triticum durum, wheat genotypes Introduction Foliar blight is the most serious biotic constraint to wheat (Triticum aestivum) yields in the rice-wheat system of South Asia. In this warm environment, foliar blight is commonly referred to as helminthosporium leaf blight (HLB), because it often occurs as a complex of spot blotch and tan spot, caused by Cochliobolus sativus and Pyrenophora tritici-repentis, respectively (Maraite et al., 1998; Duveiller & Dubin, 2002). Alternaria triticina has often been reported as part of this disease complex (Chaurasia et al., 1999, 2000). Initial alternaria leaf blight symptoms consist of small (< 1 mm), oval, yellow lesions, irregularly scattered on the leaves. As the lesions enlarge, they appear irregular and dark brown to grey, surrounded by a bright yellow margin. At a later stage, several lesions coalesce, covering large areas of the leaf and sometimes causing plant death *E-mail: mercadovergnes@fymy.ucl.ac.be Accepted 14 December 2005 © 2006 The Authors Journal compilation © 2006 BSPP (Prabhu & Prasada, 1966). Anahosur (1978) reported that A. triticina could be found on leaves, leaf sheaths, glumes and grain in Triticum spp., triticale (Triticosecale) and barley (Hordeum vulgare), whereas Prabhu & Prasada (1966) were unable to show its virulence on eight Poaceae species, including barley and oats (Avena sativa). These authors also reported clear differences in resistance and susceptibility among genotypes within the same Triticum species. Durum wheat (T. durum) genotypes such as cv. Bansi were found to be highly susceptible in eastern Uttar Pradesh, India (Prabhu & Prasada, 1966). Similarly, Casulli (1990) reported susceptibility of Italian durum wheat genotypes in southern Italy. Semidwarf bread wheat genotypes highly resistant to rust diseases were also found to harbour resistance to alternaria leaf blight (Prabhu & Prasada, 1966). However, in separate studies in India and Mexico, bread wheat genotypes RR21 (Sonalika) and Bobwhite SH9846 were used as susceptible controls (Sinha et al., 1991; Chaurasia et al., 2000; Pellegrineschi et al., 2001). Recent pathogenicity tests with A. triticina reference type isolate IMI 289962 from India showed resistance to A. triticina among bread wheat 485 486 D. Mercado Vergnes et al. genotypes resistant to HLB in Asia (Mercado Vergnes et al., 2002). Among the 15 genotypes tested, only the durum cv. Bansi used as a control was found to be susceptible. As in other Alternaria species, A. triticina produces toxic metabolites. A culture filtrate of A. triticina was reported to inhibit the germination of seeds and after 2 days induce necrotic spots irregularly scattered on the leaf lamina of 5-week-old plants (Vijaya Kumar & Rao, 1979). The toxic principle is thermostable and nonspecific (Vijaya Kumar & Rao, 1979). Fàbrega et al. (2002) reported that A. triticina isolate IMI 289962 produced tentoxin, a nonhost-specific toxin that could contribute to pathogenicity, but is not considered of primary importance during fungal penetration in plant tissues (Vijaya Kumar & Rao, 1979). In the same study, A. alternata isolate IMI 289680, incorrectly reported as A. triticina, did not produce tentoxin, but it did produce althernariol monomethyl ether, altertoxin and tenuazonic acid. The identification of Alternaria species by morphological characters requires a combination of rigorous methods. A three-dimensional sporulation pattern (length of primary conidiophores, branching types and origin of branching, conidial shapes and ornamentations) was introduced by Simmons & Roberts (1993) to facilitate the classification and identification of small-spored Alternaria isolates by microscopy. They described six major groups with characteristic sporulation patterns. Sporulation group 6 (the A. infectoria species group) consists of A. infectoria, A. oregonensis, A. triticimaculans, A. metachromatica and A. conjuncta, together with several undescribed taxa (Andersen et al., 2002). Alternaria triticina can also be considered as a rudimentary taxon for this group (Simmons, 1994), whose sporulation pattern is distinctive because of the strongly pseudorostrate nature of many of the conidia. In addition to morphological criteria, different types of molecular analysis have been used to identify and classify Alternaria species, but with variable results. Even if ITS sequencing and phylogenetic analysis do not always allow the recognition of closely related species (Andersen et al., 2002, 2005), they allow discrimination between the A. infectoria species group and other Alternaria isolates that are not associated with leaf blight of wheat (Chou & Wu, 2002; Pryor & Bigelow, 2003). Since this pathogen was first described in India (Prasada & Prabhu, 1962), many authors in South Asia have indicated that this fungus was the main cause of foliar blight occurring in the wheat-growing areas of the Gangetic plains (Kulshresta & Rao, 1976; Ram & Joshi, 1979; Raut et al., 1983; Sinha et al., 1991; Chaurasia et al., 1998; Joshi et al., 1998; Tyagi et al., 2000). Only a few limited studies from other regions (Mexico, Italy, Morocco and Germany) reported the occurrence of A. triticina, which is considered to be seed-transmitted (Frisullo, 1982; Casulli, 1990; Agarwal et al., 1993; Adame Beltran & Diaz Franco, 1997; Pellegrineschi et al., 2001; Joshi & Miedaner, 2003). Several of these studies and many other reports are compilations from other sources and lack the pathogenicity tests needed to aid the identification of A. triticina (Rotem, 1994; Singh et al., 1980, 2002). Most of the studies report only on a range of foliar blight symptoms on wheat species where various types of Alternaria have been isolated or observed to colonize the lesions. There is no published evidence that all these Alternaria isolates were correctly identified as A. triticina. Also, in several instances, including in Mexico and the Indian subcontinent, the saprotrophic A. alternata is commonly found colonizing physiological leaf spot and foliar blight lesions on wheat where C. sativus and P. tritici-repentis are suspected as causal agents of leaf spotting, but where production of conidia has not yet been observed on field samples (Duveiller & Dubin, 2002). The main objective of the present study was to identify Alternaria triticina among Alternaria species isolated from wheat leaves reported to be infected by alternaria leaf blight by comparing sporulation patterns, ITS sequences, pathogenicity assays and toxicity of culture filtrates. A second objective was to evaluate the importance of A. triticina in modern wheat cultivars from Mexico and South Asia. Materials and methods Morphological characterization of isolates Twenty-four isolates from wheat leaves showing blight lesions collected in regions where alternaria leaf blight has been reported were studied (Table 1). Single-spore isolates were cultured on potato carrot agar (PCA) to characterize conidial morphology and sporulation pattern group (Simmons, 1992). For each isolate, three 5-mm-diameter plugs were transferred onto three plates and incubated at 20 ± 1°C and 10/14 h fluorescent-light/darkness for 7 days. The sporulation pattern group of each culture was examined using a stereomicroscope (×50 magnification) following the method of Simmons & Roberts (1993). Pathogenicity trials A selection of 14 Alternaria isolates were grown on PCA for 10 days as specified above. The isolates tested included the type isolate IMI 289962 and isotype isolate IMI 178784 as references for A. triticina, and IMI 289680 as a reference isolate for A. alternata. The other isolates included A. arborescens sensu Simmons MUCL 42525, MUCL 44259, MUCL 44260, MUCL 44261 and MUCL 45333, A. alternata sensu Simmons MUCL 42372, MUCL 44262 and MUCL 45332, A. tenuissima sensu Simmons MUCL 42464 and MUCL 42561, and A. triticina sensu Simmons MUCL 42465. The isolates were obtained from wheat leaf samples collected in wheat fields in Ciudad Obregon (State of Sonora) in Mexico and in Uttar Pradesh (eastern India), where alternaria leaf blight has been reported (Adame Beltran & Diaz Franco, 1997; Chaurasia et al., 1998). To identify isolates putatively corresponding to A. triticina, wheat genotypes RR21, Bobwhite SH9846, Bansi and A-9-30-1 were selected for pathogenicity tests based on previous studies (Prasada & Prabhu, 1962; Sinha Plant Pathology (2006) 55, 485–493 Identification of Alternaria on wheat 487 Table 1 Identities, geographic origins, host species and sources of Alternaria spp. single-spore isolates used in this study Isolated a Isolate Species Geographic origin From By Date IMI 289962 IMI 178784 MUCL 42465 IMI 289680 MUCL 42320 MUCL 42372 MUCL 42458 MUCL 42459 MUCL 42464 MUCL 42475 MUCL 42480 MUCL 42481 MUCL 42525 MUCL 42560 MUCL 42561 MUCL 42562 MUCL 42563 MUCL 42564 MUCL 44259 MUCL 44260 MUCL 44261 MUCL 44262 MUCL 45332 MUCL 45333 A. triticina A. triticina A. triticina A. alternata A. alternata A. alternata A. alternata A. arborescens A. tenuissima A. arborescens A. alternata A. arborescens A. arborescens A. arborescens A. tenuissima A. tenuissima A. tenuissima A. arborescens A. arborescens A. arborescens A. arborescens A. alternata A. alternata A. arborescens Delhi, India Delhi, India Masoda, India India Kimberley, South Africa Aligard, India Basti, India Basti, India Masoda, India Pantnagar, India Ambala, India Pirsabat, Pakistan Varanasi, India Varanasi, India Varanasi, India Varanasi, India Varanasi, India Varanasi, India C. Obregón, Mexico C. Obregón, Mexico C. Obregón, Mexico El Batán, Mexico C. Obregón, Mexico Varanasi, India Triticum aestivum Triticum sp. T. durum Triticum sp. Triticum sp. Triticum sp. T. aestivum T. durum T. durum T. durum T. durum T. aestivum T. aestivum T. aestivum T. aestivum T. aestivum T. aestivum T. aestivum T. aestivum T. aestivum T. aestivum T. aestivum Triticum sp. Triticum sp. Indian Agric. Res. Institute – T. Di Zinno A. Kumar T. Di Zinno R. Chand T. Di Zinno T. Di Zinno T. Di Zinno T. Di Zinno T. Di Zinno T. Di Zinno R. Chand T. Di Zinno T. Di Zinno T. Di Zinno T. Di Zinno T. Di Zinno M. Mezzalama M. Mezzalama M. Mezzalama M. Mezzalama D. Mercado D. Mercado March, 1960 – March, 1994 November, 1984 October, 1993 May, 1993 March, 1994 March, 1994 March, 1994 March, 1994 April, 1994 April, 1994 March, 1996 March, 1996 March, 1996 March, 1996 March, 1996 March, 1996 March, 2000 March, 2000 March, 2000 March, 2002 March, 2000 March, 2002 a IMI, CABI Bioscience Genetic Resource Collection, Bakeham Lane, Egham, Surrey, UK. MUCL, BCCMTM/MUCL Mycothèque de l’Université Catholique de Louvain, Louvain-la-Neuve, Belgium. b et al., 1991; Chaurasia et al., 1999, 2000; Pellegrineschi et al., 2001; Mercado Vergnes et al., 2002) (Table 2). Plants were sown in 13 × 11 cm plastic pots (three plants per pot) containing an autoclaved 1:1 silt clay soil and compost mixture. The plants were grown in a glasshouse at 25/20°C day/night temperature with a 16 h photoperiod, watered on alternate days and supplied with 0·4 mL of a 7:5:6 NPK liquid fertilizer (Substral®; Scotts) once a fortnight. Two experiments were conducted, using plants at the four-leaf and heading stages. Both experiments were arranged following a randomized complete block design using four pots with five plants each for the pathogenicity test at the seedling stage, and two pots with five plants each for the trial on plants at the heading stage. The glasshouse experiments were repeated once. To evaluate the importance of A. triticina in modern wheat cultivars, 11 spring wheat genotypes of diverse origin (Table 2) were tested together with RR21, Bobwhite SH9846, Bansi and A-9-30-1 for their reaction to A. triticina IMI 289962, IMI 178784 and MUCL 42465. Nonpathogenic A. alternata isolates IMI 289680 and MUCL 42372 were included as controls. The experiment was conducted using plants at the four-leaf stage and arranged following a randomized complete block design using three pots with five plants each. The experiment was repeated once. Conidia were harvested by flooding the Petri plate with sterile distilled water amended with two drops of Tween 20 per 100 mL and by scraping the agar surface with a Plant Pathology (2006) 55, 485–493 Table 2 Names and origins of 15 wheat genotypes used in this study Name of genotype Origin Species Achyut A-9-30-1a Bansib Bhrikuti Bobwhite SH9846c BL 1473 BL 1887 Chirya 3 Ciano T-79 Gourab HD2329 Kanchan NL 297 PBW 373 RR21 (Sonalika)d UP262 Nepal/CIMMYT India India Nepal CIMMYT Nepal Nepal CIMMYT CIMMYT Bangladesh India Bangladesh Nepal India/CIMMYT CIMMYT India Triticum aestivum T. durum T. durum T. aestivum T. aestivum T. aestivum T. aestivum T. aestivum T. aestivum T. aestivum T. aestivum T. aestivum T. aestivum T. aestivum T. aestivum T. aestivum a Resistant to A. triticina (Prasada & Prabhu, 1962). Susceptible to A. triticina (Prasada & Prabhu, 1962). c Susceptible to A. triticina (Pellegrineschi et al., 2001). d Susceptible to A. triticina (Sinha et al., 1991; Chaurasia et al., 1999, 2000). b glass rod. After filtering through a cheesecloth, the suspension was adjusted to 20 000 conidia mL−1 using a haemocytometer. The plants were sprayed with the spore suspension using an atomizer until runoff. The inoculated plants were first moved to a mist chamber for 72 h at 21/ 488 D. Mercado Vergnes et al. 17°C day/night and with a 16 h photoperiod and 15 min fogging h−1 before being transferred to the glasshouse at 25/ 20°C. In all experiments, the second and third leaves of plants at the seedling stage and the penultimate and flag leaves of plants at the heading stage were evaluated 7 days after inoculation. Plants were rated for the presence (+) or absence (–) of 2- to 5-mm-diameter spots that were irregular in shape, dark brown to grey in colour and surrounded by a bright yellow margin. Toxin production and bioassays To test for toxin production, the same 14 isolates described above were grown on PCA until they were 4–5 cm in diameter. Ten plugs 5 mm in diameter were cut from each colony and transferred to 1 L Roux bottles containing 100 mL of autoclaved liquid Fries medium (No. 66; Dhingra & Sinclair, 1985). The bottles were incubated with a daily light period of 8 h without agitation at 20°C for 1 week. Culture filtrates were collected following Bains’ method (Bains & Tewari, 1987). Culture filtrates diluted to one-fifth strength with water were prepared and assayed for toxic activity by infiltrating leaves of two spring wheat (RR21 and Bobwhite SH9846) and two durum wheat genotypes (Bansi and A-9-30-1) at the three-leaf seedling stage with approximately 20 µL suspension using a syringe without a needle. Sterile noninoculated Fries liquid medium was used as a control. The infiltrated area limits were marked with a nontoxic felt pen before the water-soaking disappeared. Symptom development was visually assessed 5 days after leaf infiltration. The toxin bioassay was repeated once. Molecular characterization of isolates Ten 5-mm-diameter plugs of each Alternaria isolate were transferred to 250 mL autoclaved medium containing 30 g L−1 potato dextrose broth (PDB; Difco) supplemented with 0·5 mL distilled water solution containing 0·72 g Fe(NO3)3·9H2O, 0·44 g ZnSO4·7H2O and 0·2 g MnSO4·4H2O L−1 at pH 5·1, and shaken in a 500 mL Erlenmeyer flask at 24 ± 1°C under continuous light for 1 week. Mycelium was collected by vacuum filtration on a Buchner funnel through two layers of miracloth (40 µm). Fungal DNA was extracted from freshly collected mycelium with phenol-chloroform as described by Lee & Taylor (1990) and diluted to a final concentration of 10 ng µL−1 for PCR reactions. rDNA from the ITS region ( ITS1, 5·8S, ITS2) was amplified with primers ITS5 and ITS4 (White et al., 1990). Thermal cycling conditions involved an initial denaturation step at 94°C for 3 min, followed by 30 cycles of 94°C for 1·5 min, 60°C for 1·5 min and 72°C for 2 min, and a final extension step at 72°C for 10 min. PCR product purifications were carried out with the QIA quick™ PCR purification kit (Qiagen Inc.) Successful PCR reactions resulted in a single band observed on a 1·6% agarose gel. Table 3 Species, sources and GenBank accession numbers used in DNA sequence analyses Species Sourcea GenBank accession Alternaria alternata A. alternata A. alternata A. alternata A. arborescens A. arborescens A. arborescens A. arborescens A. arborescens A. tenuissima A. tenuissima A. triticina A. triticina A. triticina Exserohilum pedicillatum IMI 289680 MUCL 42372 MUCL 44262 MUCL 45332 MUCL 42525 MUCL 44259 MUCL 44260 MUCL 44261 MUCL 45333 MUCL 42464 MUCL 42561 MUCL 42465 IMI 289962 IMI 178784 EEB 1336 AY714479 AY714480 AY714482 DQ242504 AY714484 AY714481 DQ242505 DQ242506 AY714488 AY714483 AY714487 AY714478 AY714476 AY714477 AF229478 a EEB, E. E. Butler, Department of Plant Pathology, University of California, Davis, CA 95616, USA; IMI, CABI Bioscience, Genetic Resources Collection, Bakeham Lane, Surrey, UK; MUCL, BCCM™/ MUCL Mycothèque de l’Université Catholique de Louvain, Louvain-laNeuve, Belgium. The ITS regions of 14 isolates were sequenced in this study (Table 3). Sequencing reactions were performed with primers ITS5 and ITS4 using the CEQ DTCS Quick Start Kit™ (Beckman Coulter, Inc.). Chromatograms were determined with a CEQ 200XL capillary automated sequencer (Beckman Coulter, Inc.). Nucleotide sequences were compiled with Sequencher (Gene Code Corporation). DNA sequences were manually aligned using the BioEdit Sequence Alignment Editor v.6·0·7 (Isis Pharmaceuticals, Inc.). Phylogenetic analyses were performed with paup Phylogenetic Software v.4·0 β programs (Sinauer Associates, Inc.). The sequence of Exserohilum pedicillatum was included as an outgroup in all analyses. Parsimony analysis heuristic searches for the most parsimonious trees were conducted using random stepwise addition and branch swapping by tree bisection-reconnection (TBR). Sequence gaps were treated as missing data. During maximum-parsimony bootstrap analysis, 500 replicates were performed to assess the statistical support of the most parsimonious tree (Pryor & Bigelow, 2003). Results Morphological characterization of isolates Direct morphological examination of all 24 isolates on PCA at ×50 magnification resulted in sporulation groupings as obtained by Simmons & Roberts (1993) and Andersen et al. (2002). Alternaria triticina isolates IMI 289962, IMI 178784 and MUCL 42465 exhibited sporulation pattern 6, similar to isolates of the A. infectoria species group, but characterized by long (> 7 µm) intercalary secondary conidiophores. Most isolates tested in the Plant Pathology (2006) 55, 485–493 Identification of Alternaria on wheat 489 Figure 1 Reactions of wheat genotypes Bansi (1), A-9-30-1 (2), RR21 (3) and Bobwhite SH9846 (4) to infiltration of 1/5-water-diluted culture filtrates from Alternaria triticina IMI 289962, IMI 178784 and MUCL 42465, A. alternata IMI 289680, MUCL 42372, MUCL 44262 and MUCL 45332, A. arborescens MUCL 42525, MUCL 44259, MUCL 44260, MUCL 44261 and MUCL 45333, A. tenuissima MUCL 42464 and MUCL 42561, and noninoculated Fries liquid medium (control). The infiltrated area limits were marked with a nontoxic felt pen before the water-soaking disappeared. Results of the second toxin bioassay. Figure 2 Symptoms produced 7 days after inoculation with Alternaria triticina isolate MUCL 42465 on third-leaf seedlings of wheat genotypes Achyut (1), A-9-30-1 (2), Bansi (3), Bhrikuti (4), Bobwhite SH9846 (5), BL 1473 (6), BL 1887 (7), Chirya 3 (8), Ciano T-79 (9), Gourab (10), HD2329 (11), Kanchan (12), NL 297 (13), PBW373 (14), RR21 (Sonalika) 15 and UP262 (16). A-9-30-1 and Bansi are durum wheat genotypes. present study, however, showed sporulation patterns 3 and 4, typical of both the A. arborescens and A. alternata groups. Both groups were characterized by conidial chains that branched mainly from the conidial body through short (2–5 µm) secondary conidiophores. However, all A. arborescens isolates showed a long primary conidiophore (100–150 µm long). The alternation of aerial and submerged mycelium growth rings on PCA was also very characteristic of the A. arborescens species group isolates. Some isolates showed sporulation pattern 5, indicative of the A. tenuissima species group, characterized by unbranching conidial chains, borne on short (2–5 µm) primary conidiophores. Toxin production and bioassays Necrotic lesions were induced only by diluted culture filtrate of isolates IMI 289962, IMI 178787 and MUCL Plant Pathology (2006) 55, 485–493 42465 in all tested wheat genotypes (Fig. 1). No symptom development was observed on any genotype as a result of infiltration with culture filtrates of IMI 289680, MUCL 42525, MUCL 44259, MUCL 44260, MUCL 44261, MUCL 45333, MUCL 42372, MUCL 44262, MUCL 45332, MUCL 42464 and MUCL 42561, nor with noninoculated Fries liquid medium. Pathogenicity trials Leaf blight lesions became visible on leaves within 48–72 h after inoculation with A. triticina reference isolates IMI 289962 and IMI 178784, and A. triticina sensu Simmons MUCL 42465 at the four-leaf stage and at the heading stage. The initial lesions were small (< 1 mm in diameter), oval, yellow and irregularly scattered on the leaves. As the lesions aged and extended, they became irregular and dark brown to grey, surrounded by a bright yellow margin. D. Mercado Vergnes et al. 490 Figure 3 Flag leaf reaction of durum wheat cv. Bansi at heading stage 10 days after inoculation with isolates Alternaria triticina IMI 289962 (1 and 2) and A. triticina MUCL 42465 (3 and 4). Table 4 Pathogenicity of 14 single-spore isolates of Alternaria spp. at the four-leaf stage (GS14) and the heading stage (GS59) on four wheat genotypes Pathogenicity on:a A-9-30-1 Bansi Bobwhite RR21 Isolate Species GS14 GS59 GS14 GS59 GS14 GS59 GS14 GS59 IMI 289962 IMI 178784 MUCL 42465 IMI 289680 MUCL 42372 MUCL 44262 MUCL 45332 MUCL 42525 MUCL 44259 MUCL 44260 MUCL 44261 MUCL 45333 MUCL 42464 MUCL 42561 Control A. triticina A. triticina A. triticina A. alternata A. alternata A. alternata A. alternata A. arborescens A. arborescens A. arborescens A. arborescens A. arborescens A. tenuissima A. tenuissima – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – + + + – – – – – – – – – – – – + + + – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – a Plants were evaluated 7 days after inoculation for the presence (+) or absence (–) of 2- to 5-mm-diameter spots that were irregular in shape, dark brown in colour and surrounded by a bright yellow margin; A-9-30-1 and Bansi are durum wheat genotypes; Bobwhite (= Bobwhite SH9846) and RR21 are bread wheat genotypes. After 7 days the lesions consisted of 2- to 5-mm-diameter spots that were irregular in shape, dark brown in colour and surrounded by a bright yellow margin (Fig. 2). As the lesions aged and extended, they became irregular and dark brown to grey. After 10 days they coalesced and covered large (> 1 cm) areas, sometimes resulting in necrosis of the entire leaf (Fig. 3). Only A. triticina isolates IMI 289962, IMI 178784 and MUCL 42465 were pathogenic on durum wheat cv. Bansi (Table 4). No alternaria leaf blight symptoms were observed with A. alternata IMI 289680 on any of the inoculated genotypes, confirming that it was a saprotroph of wheat. In addition, none of the sensu Simmons isolates of A. arborescens, A. alternata and A. tenuissima collected from leaves and reported to cause alternaria leaf blight were pathogenic on the genotypes tested in this study. Similarly, none of the 11 non-A. triticina isolates tested in this study were pathogenic to wheat cv. Bansi or to 11 modern wheat varieties from Bangladesh, India, Nepal and Mexico. Molecular characterization of isolates Successful PCR reactions resulted in a single band observed on a 1·6% agarose gel (Fig. 4). Amplified fragments of isolates IMI 289962, IMI 178784 and MUCL 42465 were 610 –620 bp, while all the other isolates yielded bands of 590 –600 bp. Purified PCR products yielded sequences of 545–595 bp in length. Alignment of the ITS sequences with those of other Alternaria genotypes and E. pedicillatum resulted in a 552-bp region, of which 76 characters (13·8%) were variable and 37 characters (6·7%) were parsimony-informative. Four regions with numerous indels were apparent and the character alignment within these regions was variable. Among them, variable region 1 spanned characters 31–70. Within this region, a notable indel (characters 46 –70) was present in sequences from members of the A. triticina species group, but not in sequences from any other isolate. Maximum-parsimony analysis of the ITS dataset yielded 100 equally parsimonious trees, one of which is shown in Plant Pathology (2006) 55, 485–493 Identification of Alternaria on wheat 491 Figure 4 PCR amplification products from the ITS region (ITS1, 5·8S, ITS2) of 14 Alternaria spp. isolates. Lane M, molecular size marker (100-bp DNA ladder); lanes 1–3, A. triticina isolates IMI 289962, IMI 178784 and MUCL 42465; lanes 4–7, A. alternata IMI 289680, MUCL 42372, MUCL 44262 and MUCL 45332; lanes 8–12, A. arborescens MUCL 42525, MUCL 44259, MUCL 44260, MUCL 44261 and MUCL 45333; lanes 13–14, A. tenuissima MUCL 42464 and MUCL 42561. tenuissima. This group was further subdivided into three clades weakly supported by the bootstrap analysis (69, 62 and 60%). Discussion Figure 5 One of the 100 most parsimonious trees from maximumparsimony analysis of ITS1/5·8S/ITS2 sequences from Alternaria and related species. Sequences determined in the course of this study appear in bold. Horizontal branch lengths are proportional to the number of nucleotide substitutions inferred to have occurred along a particular branch of the tree. Values associated with branches indicate the degree of bootstrap support expressed as the percentage of 500 bootstrapped trees in which the corresponding clades are present. Exserohilum pedicillatum was treated as an outgroup. Fig. 5. Two clades were evident and corresponded to wheat-pathogenic and nonpathogenic groups. The two clades were strongly supported by bootstrap values of 100%. The pathogenic group included the two reference isolates of A. triticina and the A. triticina sensu Simmons isolate MUCL 42465. The nonpathogenic group included A. alternata reference isolate IMI 289680 and sensu Simmons isolates of A. alternata, A. arborescens and A. Plant Pathology (2006) 55, 485–493 The pathogenicity of A. triticina, A. alternata, A. arborescens and A. tenuissima isolates from diverse geographic origins was evaluated on four wheat genotypes under controlled conditions in a glasshouse. These conditions allowed a reliable assessment to be made of the pathogenicity of A. triticina isolates on wheat and the observations consistently agreed with the results of inoculation with A. triticina reference type isolates IMI 289962 and IMI 178784. Symptoms of alternaria leaf blight were induced indistinctly after 24 or 72 h incubation periods under high relative humidity. Plants of cv. Bansi were highly susceptible at the four-leaf seedling stage and at the heading stage. Bansi, an old Indian durum wheat cultivar, was reported as susceptible to A. triticina in the 1960s (Prabhu & Prasada, 1966). Wheat genotypes RR21 and Bobwhite SH9846, reported previously as susceptible (Sinha et al., 1991; Chaurasia et al., 1999, 2000; Pellegrineschi et al., 2001), were found to be highly resistant to all A. triticina isolates used in this study. Alternaria alternata IMI 289680, MUCL 42372, MUCL 44262 and MUCL 45332, A. arborescens MUCL 42525, MUCL 44259, MUCL 44260, MUCL 44261 and MUCL 45333, and A. tenuissima MUCL 42464 and MUCL 42561 were found to be nonpathogenic on wheat, despite being collected from wheat samples showing leaf blight lesions apparently induced neither by the tan spot nor by the spot blotch pathogens, but alleged to be caused by A. triticina. The high recovery of Alternaria spp. from wheat showing blight lesions but not colonized by the tan spot or by the spot blotch pathogens could possibly be explained by the ability of Alternaria species to grow saprotrophically, as also observed on physiological leaf spot of wheat in Oregon, USA (Smiley et al., 1993). The identification of Alternaria species based on morphological criteria of the colonies and spores remains a challenging task (Maraite et al., 1998). Nevertheless, Simmons and Roberts’ sporulation pattern 6 and a restricted number of conidia per sporulation unit (two to three conidia) associated with primary conidia and long 492 D. Mercado Vergnes et al. (> 7 µm) apical secondary conidiophores growing on 7-day-old PCA were useful criteria to differentiate A. triticina isolates from the other nonpathogenic Alternaria isolates used in this study. This was the case for the recently isolated MUCL 42465, the only isolate to show sporulation pattern 6 similar to the IMI reference isolates. Interestingly, MUCL 42465 was isolated in the 1990s from leaf samples collected in durum wheat fields in Uttar Pradesh from the same durum cultivar, Bansi. Bioassays for toxic activity by leaf infiltration of seedlings at the three-leaf stage revealed that only A. triticina isolates IMI 289962, IMI 178784 and MUCL 42465 were able to induce necrotic symptoms on wheat genotypes. The toxic principle was nonspecific, as previously reported (Tyagi et al., 2000), and symptoms could be related to tentoxin, a phytotoxic metabolite produced by A. triticina IMI 289962, but not by A. alternata isolate IMI 289680 (Fàbrega et al., 2002). The phytotoxicity of culture filtrates also appeared to be a useful criterion to differentiate A. triticina from nonpathogenic A. alternata isolates. In this study, the molecular approach proved helpful in discriminating between wheat-pathogenic and nonpathogenic Alternaria isolates. Alternaria triticina IMI 289962, IMI 178784 and MUCL 42465 yielded longer ITS sequences than isolates belonging to the A. alternata species group. Thus, differences between these groups were already visible after the migration of PCR reaction products on a 1·6% agarose gel. Maximum-parsimony analysis revealed relationships that were consistent with previous studies; specifically, that A. triticina (infectoria group) composed a strongly supported clade that did not cluster within the nonpathogenic clade formed by isolates of A. alternata, A. tenuissima and A. arborescens (alternata group) (Pryor & Bigelow, 2003). However, the ITS marker could not distinguish the other Alternaria species analysed and more markers, such as the mitochondrial small subunit (mt SSU) or the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene used by Pryor & Bigelow (2003), are needed. Results suggest that most isolates obtained in recent years from wheat samples collected in the Indian subcontinent and Mexico are nonpathogenic to wheat and belong to the A. tenuissima, A. alternata or A. arborescens species. Results from this study and previous work (Mercado Vergnes et al., 2002) also suggest that A. triticina has a restricted host range among bread wheat cultivars. A possible reason for the low rate of confirmed isolation of A. triticina in recent years compared with the early 1960s may be the drastic change in wheat varieties that followed the adoption of modern cultivars after the ‘green revolution’ in the eastern plains of the Indian subcontinent. Tall wheat genotypes, susceptible to leaf blight, were replaced and the use of traditional durum wheat became very limited in this area. Foliar blight has traditionally always been considered important in this region and A. triticina was considered to be its major cause until the late 1980s (Joshi et al., 1998). However, although there have been numerous studies, very few have been accompanied by systematic pathogenicity tests confirming the virulence of observed Alternaria on wheat (Kulshresta & Rao, 1976; Singh et al., 1980, 2002; Casulli, 1990; Adame Beltran & Diaz Franco, 1997; Chaurasia et al., 2000; Joshi & Miedaner, 2003). The need to use the correct susceptible control, as shown by the vulnerability of durum wheat cv. Bansi, also confirms that A. triticina is a weak wheat pathogen. This pathogen is currently much less important than C. sativus as a causal agent of foliar blight in the Gangetic plains (Duveiller et al., 2005). Similarly, reports of the incidence of A. triticina in Germany, Morocco, Italy and Mexico should be confirmed by pathogenicity tests using the same standard susceptible genotypes; this is critical to appraise its prevalence and importance accurately, including its status as a seedborne pathogen (Wiese, 1977; Agarwal et al., 1993). Recently, Pellegrineschi et al. (2001) suggested that resistance to alternaria leaf blight in transgenic wheat plants might be increased by the antifungal activity of thaumatin-like proteins from barley, a nonhost of A. triticina. However, these preliminary observations should be confirmed with regard to the effect of physiological leaf spot on wheat genotype Bobwhite SH9846, since this genotype was found to be resistant to A. triticina in the present study. Because of the extremely frequent occurrence of saprotrophs such as A. alternata in preliminary observations of diseased samples under the microscope in the laboratory, irrespective of geographical area, this study underlines the importance of testing the pathogenicity of putative isolates under controlled conditions. Comparison with several reference isolates of the pathogen and susceptible host cultivars is important before claiming that any Alternaria isolate is A. triticina. 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