Mycopathologia 116: 29-35, 1991. �9 1991 Kluwer Academic Publishers. Printed in the Netherlands.

Identity of Pyrenophora isolates causing net-type and spot-type lesions on barley

D.B. Scott Grain Crops Research Institute, South African Department of Agricultural Development, Private Bag X29, Bethlehem 9700, South Africa

Received 23 November 1990; accepted in revised form I3 March 1991

Key words: Hordeum vulgare, Pyrenophora japonica, Pyrenophora teres

Abstract

Causative fungi of net-type and spot-type lesions on barley were isolated and identified as anamorphs of Pyrenophora teres and Pyrenophora japonica respectively. Comparison of morphological characteristics showed that isolates of the two species are separable on the basis of differences in colony texture, conidiophore shape, conidial arrangement and conidial dimensions. The two species could also be distinguished from each other on account of adult-plant reactions exhibited by Dampier barley after inoculation by various isolates from South Africa, one isolate from Germany and one isolate from Washington State in North America.

Introduction

Pyrenophora teres Drechs., teleomorph of Drech- slera teres (Sacc.) Shoem., is the cause of net blotch in barley (Hordeum vulgare L. emend. Bowden). Spot-type lesions caused by a similar fungus have also been reported on barley. McDonald [1] described two isolates, one from Canada and the other from Israel, that produced the spot-type lesion on barley. According to McDonald [1], the two isolates resembled Pyren- opora japonica Ito & Kurib., teleomorph of DrechsIera tuberosa (Atk.) Schoem., but one iso- late crossed with a known isolate of P. teres and for this reason he considered the P. japonica-type of isolates as mutant strains of P. teres.

In Denmark the spot-producing fungus was de-

signated P. teres forma maculata Smedegaard- Petersen, because Smedegaard-Petersen [2] was not able to observe morphological differences be- tween isolates causing spot-type lesions and iso- lates causing net-type lesions. More recently the spot form of net blotch was reported in Australia [3] and also in the United States [4].

In South Africa spotted lesions are common on commercial cultivars grown for malt production in the Western Cape Province. Netted lesions on barley have been reported in the past [5], but presently such lesions are observed mainly on introductions used in breeding trials. The purpose of this study was to identify causative fungi of net- and spot-type lesions on barley and to extend information regarding their morphological, taxo- nomical and pathological characteristics.

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Materials and methods

Collection and isolation of fungi. The isolates used in this study were recovered from spotted and netted lesions on leaves of different barley genotypes. Most of the leaves were collected in 1989 during inspections of breeding trials in the Rfiens area of the Western Cape Province.

Leaf specimens were placed in envelopes and allowed to dry before individual lesions were cut out and cultured on water agar (WA) containing 20 mg ~ streptomycin and 10 mg 1 benomyl. Cul- tures were incubated at 17 ~ in the dark and 5 d later single conidia from conidiophores de- veloping close to the edge of each lesion were transferred to WA.

Isolates selected for cultural studies and patho- genicity tests carried the designations PREM 50529--PREM 50546. One isolate, PREM 50544, derived from leaf spots on wild barley (Hordeum murinum L.), which is a common weed in the barley-producing areas of the Western Cape Province. Cultures representing an isolate from net-type lesions on barley in the United States, PREM 50537, and an isolate from spot-type le- sions on barley in Germany, PREM 50546, were included for comparison.

Cultural studies. Barley isolates of Pyrenophora can vary greatly with respect to colony morpho- logy and sporulation, especially after successive subculturing [6]. To avoid such variation, taxo- nomic characteristics were recorded as far as pos- sible from cultures representing wild-type iso- lates.

Pyrenophora isolates from barley usually do not sporulate easily on common laboratory media. Previous studies showed that sporulation of barley isolates is induced by light or uv irradi- ation on media containing carbendazim-gen- erating fungicides [1, 7, 8]. The method adopted in this study was as followed: Single-spored cul- tures were grown on WA in darkness at 20 ~ After 10 d, when colonies had filled 9 cm diam plates, cultures were transferred aseptically with a stainless-steel spatula onto sucrose asparagine

agar (SAA) containing distilled water 1 L; suc- rose, 2.5g; asparagin, 0.27g; KH2PO4, 0.15g; K2HPO4, 0.15 g; MgSO4.7HaO, 0.01 g; benomyl, 0.01 g and Difco agar 15 g. The cultures were first incubated at 20 ~ for 2 d under mixed daylight- type and near ultra-violet lamps (Philips RSF 40 BLB) and thereafter placed in darkness at 17 ~

Artificially infected leaves were used for in- vestigating the morphology of conidia and conidi- ophores. Dried leaves of barley plants (cultivar Dampier) were cut into 3 cm pieces, wetted in 95% ethanol for 10 sec and surface-sterilised in 1% NaOC1 solution for 90 sec. This tissue was then rinsed in sterile water, blotted on tissue paper to remove excess water and transferred to petri dishes containing Whatman filter paper saturated with sterile water. Small plugs, 0.5 cm in diam, from WA cultures of the isolated fungi were transferred aseptically and separately onto the sterilised leaves in each petri dish. Cultures received natural light on a laboratory bench at 20-25~ Sporulating structures were mounted in lactophenol and examined under a standard research microscope.

Agar cultures, infected leaves and incubated host material were dried and deposited in the National Fungus Collection (PREM), Plant Pro- tection Research Institute, Pretoria.

Pathogenicity tests. Inoculum for pathogenicity tests was prepared by flooding sporulating cul- tures on SAA with sterile distilled water to which Tween 20 surfactant was added at the rate of one drop per 100cm. Spore suspensions were ob- tained by gently scraping the surface of a culture with a rubber spatula. The number of spores in the suspension was adjusted to a concentration of approximately 5 x 103 per cm. Spore suspensions of each isolate were atomised onto adult plants of Dampier and Clipper barley. Single plants of the two cultivars were planted separately in 15 cm diam pots and grown in a greenhouse under stan- dard conditions of 20 ~ day/15 ~ night with a photoperiod of approximately 12 h. Inoculated plants were placed in a dark mist chamber at 25 ~ for 24 h after which they were returned

Table 1. Differences in cultural and morphological features of Group A and Group B isolates of Pyrenophora.

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Characteristic Group A isolates Group B isolates features (PREM 50529- (PREM 50538-

PREM 50537) PREM 50546)

Sporulation Abundant Sparse Colony texture Granular Floccose Pycnidia Absent Present Secondary conidiophores Sparse Abundant Apex of conidiophore Broad Thin Conidial arrangement Often catanulate Never catenulate Length of cnnidia 40-120 (150) ixm 50-145 (210) ~m

back to the greenhouse. Inoculated plants were examined periodically for type of lesions which developed on penultimate leaves. Infected leaves were plated out on WA for reisolation of original isolates.

Results

Isolates could readily be separated in two groups on the basis of colony morphology, characteristics of sporulating structures and type of foliar lesions produced on headed barley plants. Characteristic features of Group A and Group B isolates are summarized in Table 1.

On SAA Group A isolates produced abundant conidia among minute droplets of clear exudate. Colonies had a granular appearance due to abun- dant sporulation and the development of scleroti- urn-like bodies or protothecia projecting from the agar surface. Protothecia were setose, globose, 300-600 p,m in diam, composed of isodiametric cells with thickened walls. The colony reverse showed uniform brown or black colours with prominent dark-brown hyphae radiating through- out the agar. Conidiophores were simple, erect, up to 200 Ixm long on infected leaves, but usually shorter, less than 50 txm long when arising from aerial hyphae in agar cultures (Fig. la). Each conidiophore typically showed a broad apex on which prominent scars occurred on closely situ-

ated bends. Conidia developed singly or in chains on the apical part of each conidiophore (Fig. lb). The conidia, measuring 40-120 • 19-21 ixm, or occasionally up to 150 txm long, were yellowish brown, cylindrical or flask shaped with broad hemispherical ends, widest near the middle or at the inflated basal cell. Conidial cells were mostly longer than wide and sometimes constricted at the septa.

On SAA Group B isolates formed sparsely spo- rulating, dark-grey colonies covered with a fair amount of aerial hyphae. Submerged growth con- sisted of dark radiating hyphae bearing pycnidia and protothecia. Pycnidia were olivaceous brown, globose to pyriform, about 150 Ixm in diam, con- taining hyaline, aseptate, ovoid or cylindrical, non-germinating spores, 1.5 • 3.0 • 1-2 ixm. Conidiophores were reddish brown, erect, sept- ate, mostly unbranched with thick walls and up to 400 txm long. The upper part of the conidiophore assumed a zig-zag pattern with conidiogenous nodes spaced far apart from each other (Figs. lb, lc). Conidia were olivaceous brown, rounded at both ends, but with the basal cell showing a prominent scar, cylindrical, but slightly tapering towards the basal cell in some cases (Figs. lc, ld). Fully developed conidia were mostly 4 -6 septate, 50-145 • 16-20 txm, but in some isolates up to 210 ixm long. Many conidia produced sec- ondary conidiophores at one or both end cells under normal conditions.

Isolates in both groups tend to become atypical

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Fig. 1. Conidia and conidiophores of Pyrenophora teres and Pyrenophora japonica, • 400. (a) Conidiophores of P. teres arising from aerial hyphae, showing broad apices with prominent scars on closely situated bends; (b) Conidia of P. teres arranged singly and alternatively at the fertile apex of the conidiophore;(c) Primary conidiophores of P. japonica; (d) Upper part of primary conidiophore and detached conidia of P. japonica; (e) Fully developed conidia of P. japonica.

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Fig. 2. Symptoms elicited by Pyrenophora teres on leaves of two barley cultivars. Left to right: Dampier one week after inoculation, Dampier two weeks after inoculation, Clipper one week after inoculation, Clipper two weeks after inoculation.

Fig. 3. Symptoms elicited by Pyrenophora japonica on leaves of two barley cultivars. Left to right: Dampier one week after inoculation, Dampier two weeks after inoculation, Clipper one week after inoculation, Clipper two weeks after inoculation.

after a few transfers on agar media. Tufts of hy- phae and sterile mycelium were produced on media rich in carbohydrates, It was impossible to identify isolates after cultures have degenerated, because conidia were often lacking in such cul- tures or if present they showed abnormal shapes and dimensions.

The two groups of isolates separated on ac- count of morphological characteristics could clearly be distinguished in pathogenicity tests. On the cultivar Dampier, Group A isolates pro- duced typical net-blotch lesions, having dark- brown striations extending both longitudinally and transversely within the lesion to form a clear net-like pattern (Fig. 2). Isolates were extremely virulent on this cultivar and lesions expanded rap- idly, causing severe leaf necrosis over the entire leaf blade during the test period. Clipper barley on the other hand showed a high degree of resis- tance to Group A isolates. Most isolates failed to

produce lesions on this cultivar, but a few pro- duced pin-point lesions which remained restricted in size (Fig. 2). Group B isolates produced dark- brown spots on both cultivars. Lesions ranged from small circular spots to large elliptical spots with varying amounts of chlorosis around each spot (Fig. 3), Lesions often coalesced when their numbers were high, but they never resulted in death of the entire leaf blade.

Discussion

Comparative morphological studies showed that the Pyrenophora isolates causing net-type and spot-type lesions on barley represent two species, which according to descriptions given by Sivane- san [9], should be named P. teres and P. japonica

respectively. Both species are parasites of culti-

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vated barley, but during this study P. japonica was also found on wild barley in South Africa.

Schoemaker [10] was the first to recognize P. japonica as a cause of leaf spots on barley. The original description of P. japonica was based on ascomata that occurred on half rotten straw or stubble in Japan. Previously Schoemaker [11] showed that the anamorph of P. secalis White- head & Dickson is conspecific with D. tuberosa and that P. secal& should be treated as a synonym of P. japonica. Although P. japonica has many characteristics in common with P. teres, Schoe- maker [10] pointed out important differences be- tween the two species. For instance, while P. teres is known primarily as a parasite of barley, P. japonica occurs on barley as well as on other cereals and wild grasses. Futhermore, in P. japon- ica fully developed conidia are longer than those of P. teres, they lack the inflated basal cell and regularly have secondary conidiophores. In an- other study catanulation of conidia has been ob- served in P. teres, but not in P. japonica [6]. Present investigations confirm these morpho- logical differences between the two species. It was also found that isolates of P. japonica consistently produced pycnidia when cultured on media as used in this study. Such structures were not ob- served in cultures of P. teres. The pycnidial bodies contained spores that did not germinate, and it is quite possible that they actually represent sperm- atia, but no prove could be obtained that they definitely have the function of spermatia. Pycni- dia have previously been described for P. teres, but not for P. japonica [9].

Similar types of lesions were obtained by iso- lates of P. japonica on adult plants of Dampier and Clipper barley. On both cultivars lesions spread linearly on leaves, but even with the most virulent isolate there were limited amounts of chlorosis or necrosis in the surrounding tissues. In Australia an intermediate reaction was obtained when spot-type isolates of Pyrenophora from Australia and Canada were used to inoculate seedlings of Dampier and Clipper barley [3]. Similarities in virulence patterns among isolates

from different continents indicate a high degree of stability in natural populations of P. japonica. Arguments in favour of rejection of P. japonica on account of its interfertility with the net-blotch pathogen as reported by McDonald [1] can not be accepted. According to Smedegaard-Peterson [12] isolates of P. teres will also cross with isolates of P. grarninea Ito & Kurib. to produce hybrids with symptoms different from those caused by the parental species. Hybridisation under natural conditions seems unlikely because typical ex- amples of leaf stripe still occurs on barley in many parts of the world. The latter disease has been reported in South Africa [5], but in recent years it seems to have disappeared, probably because the fungus in this country has been eradicated by seed treatments.

Resistance to P. fetes is recognize by a reduc- tion in lesion size [13]. In Clipper barley lesions were lacking or so small that this cultivar obvi- ously should be regarded as highly resistant to net blotch. In Australia, Clipper has been de- scribed as field susceptible to spot-type isolates, but resistant to net-type isolates of Drechslera [3]. The highly susceptible reactions obtained on Dampier confirms previous reports that this culti- vat has no field resistance against net blotch [14].

Bockelman et al. [4] remarked that isolates from spot symptoms on barley produced net symptoms when inoculated into seedlings. This could well be the case if isolates were made from resistant cultivars. It is clear from previous and present investigations that field symptoms can not be used to distinguish between infections caused by P. teres and P. japonica. Spot-type net blotch caused by P. japonica might also be confused with spot blotch caused by Cochliobolus sativus (Ito & Kurib.) Drechs. ex Dast., teleomorph of Bi- polaris sorokiniana (Sacc.) Shoem. Moreover, there are non-parasitic diseases, such as leaf spots caused by boron toxicity, which closely resemble fungal leaf spots. When dealing with unknown cultivars in strange localities it would be advisable to verify disease agents by isolation and taxo- nomic studies.

Acknowledgements

Professor GW Bruehl of Washington State Uni- versity in Pullman, USA and Dr M Baumer of the Bayerische Landesanstalt ftir Bodenkultur und Pttanzenbau in Freising, Germany, supplied barley leaves from which isolates of P. teres and P. japonica were obtained during a study tour in 1989. This research was supported in part by the South African Wheat Board.

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