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Morphological and host range studies of Melampsora rusts attacking Salix species in New Zealand
1163
Mycol. Res. 100 (10): 1163-1175 (1996) Printed in Great Britain
Morphological and host range studies of Melampsora rusts attacking Salix species in New Zealand
A. G. SPIERS A N D D . H. HOPCROFT The Horticulfure and Food Research Institute of New Zealand Lfd, Palmersfon North, New Zealand
Comparative studies of Melampsora rusts pathogenic to willows in New Zealand showed that recognition of two named species was possible. These were M . coleosporioides, attacking tree willows, particularly S. babylonica and M . epifea var. epitea (according to Wilson & Henderson), and M . brici-epifea f. sp. larici-epifea typica (according to Gaumann), which was pathogenic to S. cinereal5 caprea with Larix as an alternate host. Two other Melampsora msts pathogenic to S. viminalis and S. daphnoidesls. incana x open pollinated hybrids respectively were not identifiable to species level from published descriptions. Useful taxonomic criteria included morphological features of urediniospores (shape, dimensions, ratios, wall-thickness, germpores), dimensions of paraphyses heads, teliospores, metabasidia and basidiospores. A plea is made for revision of willow rust taxonomy and for more detailed rust descriptions.
Willows have been extensively planted throughout New Zealand since their introduction in the 1840s. They have been utilized for amenity plantings, i.e. S. babylonica (weeping willow), S. alba var. vitellina pendula (golden weeping), 5. x sepulcralis ( S . babylonica x S. alba), S. matsudana (Peking willow), S. mafsudana cv. Tortuosa (corkscrew willow), S. alba var. vitellina (golden willow), S. incana, S. purpurea, S. viminalis, S. matsudana x S. alba (hybrid willows); for soil conservation, i.e. S. mafsudana, S. mafsudana x S. alba and horticultural shelter, i.e. S. matsudana, S. matsudana x S. alba and S. x caprea (pussy willow). The osier willows, S. purpurea, S. triandra and S. viminalis have also been utilized to a limited extent for basketware (Van Kraayenoord, 1975). Leaf rust caused by Melampsora species is the most important growth-limiting disease afflicting willows in New Zealand. Extensive monoclonal planting of a few select cultivars is being avoided by planting cultivar mixture and having a number of genetically diverse cultivars available. The vulnerability of monoclonal plantings to rust in New Zealand was clearly demonstrated by the impact of Melampsora laricipopulina and M . medusae on poplar plantations (Van Kraayenoord et al., 1974). In Canada, England and West European countries, willows (S. viminalis clones and other Salix spp.) have been grown for renewable energy (Ager ef al., 1986; Venvijst, 1990; McCracken & Dawson, 1992). Foliar rust caused by Melampsora species has been a major problem, causing premature defoliation and markedly reduced yields (McCracken & Dawson, 1992) which has highlighted the problems of willow rust identification (Dawson, 1988; Ronnberg-Wastljung & Gunnerbeck, 1985; Pei et. al., 1993, 1995). There is much confusion regarding the identity of
Melampsora species attacking willows in New Zealand. There appear to be four morphologically distinct rusts. Two of these, M. epifea Thiim. and M . coleosporioides Dietel, have been briefly described by Dingley (1977) and Latch (1980) respectively, while the two other rusts attacking cultivars of S. viminalis and S. daphnoides have been briefly described by Spiers & Hathaway (1987). In this paper the morphology and host range of Melampsora species attacking willows in New Zealand are described.
MATERIALS A N D M E T H O D S Host range and susceptibility A willow cultivar collection held as stools (five stools per cultivar) at Aokautere, Palmerston North was rated for Melampsora infection during March and April each year. Monitoring commenced in 1977 and is continuing. Rust severity was assessed using the Schreiner-van der Meiden rating system (Schreiner, 1959; van der Meiden, 1961). Host records were confirmed by microscopic measurement of uredinial features.
Host inoculation Inoculations of detached leaves (Table 5) were conducted to clarify the host ranges of M. epitea and the rusts attacking S. viminalis and S. daphnoidesl.5. incana x open pollinated cultivars. Inoculations were made in December when fresh urediniospores and rust-free leaves were available. Leaf discs (20 mm diam.) and small leaves were inoculated abaxially by brushing on urediniospores. Discs and leaves
Studies of Melampsora rusts
1164
Table 1. Host range, susceptibility and uredinial dimensions of Melampsora epitea from shrub willows Urediniospore dimensions ( p m ) Rust rating (March-April)
Accession number
S. hippophaefolia S. cinerea cv. Tricolor Mac 5. incana discolor S. aurita var. philicifolia S. cinerea oleifolia S. cinerea 'M' S. cinerea 'W' S. silesiaca ( x calodendron) 5. caprea x uiminalis cv. Balana 5. caprea x uiminalis 'SM' S. caprea x uiminalis 'SCCB' 680 S. dichroa (aurita x purpurea) 367 S. seringeana 667 S. purpurea cv. Pyramidalis 309 S. caprea F (5. x reichardtii) S. purpurea x uiminalis Populifera 301 332 5. pontederana x gracilisytla 215 S. caprea 'PG' (discolor B) 229 5. medemi (aegyptiaca) 306 S. caprea x uiminalis 'Hybrida' 714 S. mwcina 237 5. pontederana 346 S. pip0 12 347 S. pip0 13 S. osier cv. Schmidt 628 Mean urediniospore dimensions Coefficient of variation (CV%)%
Length
Breadth
14.9 15.2 15.6 16.7 15.9 16.3 15.5 14.9 14.7 15.8 14.9 15.5 15.3 15.3 15.6 15.6 16.3 16.9
13.0 13.0 13.1 12.5 14.4 13.6 12.6 12.6 12.7 14.3 13.0 13.3 13.1 126 13.6 13.4 13.7 13.5
Wall thickness
224 324
Not Not Not Not
measured measured measured measured
15.5 3.8
13'1 4.2
Rust rating = Schreiner-van der Meiden rating system. 2
C V % coefficient of variation = standard deviation (100). mean
Table 2. Host range, susceptibility and urediniospore dimensions of Melarnpsora epitea from S. incana cultivars Urediniospore dimensions ( p m ) Accession number
Rust rating (March-April)
Length
Breadth
Wall thickness
S. daphnoides var. acutifolia S. daphnoides ' G ' S. incana x open pollinated S. incana x open pollinated S. incana x open pollinated S. incana x open pollinated S. incana x open pollinated S. incana x acutifolia S. incana x acutifolia S. incana x acutifolia 5. incana x acutifolia 5. incana x acufifolia Mean urediniospore dimensions Coefficient of variation (CV%)2 331 213 NZ1005 NZ1006 NZ1008 NZ1009 NZlOl2 NZ1228 NZ1230 NZ1237 NZ1226 NZ1227
Rust rating = Schreiner-van der Meiden rating system. 2
CV%
of variation = standard
mean
(100)
were incubated on moistened filter paper under natural light at 20 OC. Following 7, 10 and 15 d incubations, discs were examined for erumpment uredinia. Identification of Melampsora rusts was confirmed by microscopic examination of urediniospores.
Rust morphology
Rust samples were collected from willows growing at Aokautere during March (Tables 1-4). For each collection, the host species and gross features of uredinia and telia (when
A. G. Spiers and D. H. Hopcroft
1165
Table 3. Host range, susceptibility and urediniospore dimensions of Melampsora coleosporioides from shrub willows Urediniospore dimensions (pm) Accession number
Rust rating (March-April)
S. babylonica x humboldtiana '160-16' S. argentinensis cv. Mestizo Pereyra S. babylonica S. babylonica var. annularis 5.babylonica x fragilis cv. Blanda S. babylonica x fragilis 5.babylonica x fragilis S. elegantissima (babylonica x fragilis) S. fragilis hybrid cv. Benmore S. pendulina (babylonica xfragilis) S. chilensis S. matsudana tortuosa 'NG' 5.pentandra 'G' S. penetandra cv. Dark French S. pentandra cv. Patent Lumley S. matsudana 'LG105' S. matsudana pendula 'CHI' S. matsudana ' CH2 ' S. matsudana war. turiuosa cv. Northland A S. matsudana var. tortuosa cv. Northland B S. matsudana cv. Nelson purple S. meyeriana S. salamonii S. sepulchralis (babylonica x alba) S. matsudana var. tortuosa S. matsudana 'CH4' S. babylonica x alba '131-25 ' S. babylonica x alba '131-27' S. matsudana 'YN 102' S. matsudana 'YN 207' S. matsudana 'CH 3B' S. matsudana cv. Nelson green S. coerulea 'M' S. matsudana 'CH3A' S. himalayas s.fragiiis '9/17' S. fragilis Illectic V 10/30 25 9 S. elegantissima (babylonica x fragilis) Mean urediniospore dimensions 661 658 292 321 217 277 311
(CV%)4
Length
Breadth
19.7 21.4 205 21.5 20.8 21.4 19.6 20.9 19.6 20.3 207 20.8 20.0 21.0 20.9 21.0 20.9 21.5 206 21.4 202
13.7 13.3 13.3 13.9 12.8 13.2 13.8 13.4 14.3 13.2 13.5 14.3 13.9 13.3 13.7 13.6 14.1 13.9 14.5 14.7 13.7
Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not 20.7 2.9
Wall thickness
formed formed formed formed formed formed formed formed formed formed formed formed formed formed formed formed formed 13.7 3.6
Rust rating = Schreiner-van der Meiden rating system. S = fine yellow or black spots. T = fine telia formed. 4
CV% coefficient of variation
= standard
mean
(100).
formed) were recorded. Urediniospores were mounted in lactophenol and stained with 0.5% acid fuchsin. Sixty uredinospores per collection were measured using a Leitz micrometer (under interference contrast) at 5 0 0 ~ magnification. Dimensions recorded were urediniospore length, breadth and wall thickness. From these, ratios of length to breadth (L:B); length to wall thickness (L:W); length by breadth (L x B) and length by breadth over wall thickness (L x B/W) were calculated. Urediniospore dimensions and ratios were analysed by the SAS procedure GLM (Freund & Littell, 1981). The length and breadth dimensions and wall thickness of paraphyses heads were measured. Also, echinulation and presence of germpores in urediniospores was recorded. Sixteen
collections of Melampsora (four of each possible species) from the Aokasutere herbarium were also examined. Urediniospore dimensions and ratios were measured and calculated as detailed above for field collections. To determine the morphology and location of uredinia and telia, small leaf sections (1x 5 mm) were prepared for transmission electron microscopy as previously described (Spiers & Hopcroft, 1985). Urediniospore morphology was examined by scanning electron microscopy (Spiers& Hopcroft, 1985). During late autumn (April-May) leaves bearing teliospores of M. epifea, M . coleosporioides and the Melampsora infecting 5. viminalis were collected and stored dry, in the dark at zO,for 5 months. To induce telial germination the leaves were
Studies of Melampsora rusts
1166
Table 4. Host range, susceptibility and urediniospore dimensions of Melampsora sp. from S. viminalis cultivars Urediniospore dimensions (vm) Accession number
Rust rating (March-April)
Length
Breadth
Wall thickness
S. purpurea x viminalis cv. Populifera S.inferior 'F ' S. purpurea x viminalis cv. Harrison S. viminalis 'NCCB' S.viminalis cv. Praecox S. viminalis cv. Black Osier S. viminalis cv. Stone Osier S.viminalis 'M' S. purpurea x viminalis sessifolia S. purpurea pyramidalis S. viminalis cv. Gigantea S. viminalis cv. Bowles hybrid S. pontederana x gracilis S. incana x discolor S. caprea x viminalis 'SM' S.caprea ' N ' S. pentederana S. hippophuefolia 'Kew' S. hippophaefolia (ex mollisima var. trevirani) 204 S. aurita var. philicifolia S. x reichardtii (5.caprea F) 215 234 S. nigricans fosteriana 668 5. burjatica 'Germany' 634 S.walsfeiniana 609 S. purpurea cv. Green Dicks Mean urediniospore dimensions (CV%)2
375' 3 75 375 3 75 3 75 3 75 250 250 250 125 125 125 50-75 25 25 10-25 5 5 5
15.6 15.9 15.9 15.4 16.8 14.9 15.5 174 16.8 18.5 15.4 18.5 15.8 16.1 15.7 14.7 15.5 15'4 15.1
13.4 13-4 13.7 13.3 12.9 13.2 13.3 13.5 12.9 13.6 12.9 13.9 13.5 13.8 12.8 12.8 13.3 13.4 13.5
1.6 1-4 1-4 1.4 1.4 1.4 1.3 1.5 1.5 1.5 1.3 1.5 1.4 1.4 1.4 1-4 1.5 1.5 1.4
5 5 4 4
16.4 15.8 16.0 15.6 15.6 15.3 16.0 6.0
13.6 13.6 13.4 13.4 13.6 13.6 13.4 3.0
1.5 1.4 1.4 1.5 1-5 1-4 1.4 5.0
301 687 607 245 272 3 75 379 275 305 667 220 312 332 1244 303 233 237 383 224
4
2
See footnotes, Table I.
rehydrated and placed outside among year-old plants of Larix decidua, in separate enclosures. Dimensions of metabasidia and basidiospores were measured.
RESULTS Hosf range and susceptibility
A single Melampsom species was observed on Salix when observations commenced in 1977. This species was identified as Melampsora epitea (Dingley, 1977) and was found to have Larix decidua and L. leptolepis as alternate hosts (Spiers & Hopcroft, 1985).Table 1lists the host range and susceptibility of shrub willows to M. epitea. During 1978, rust infection of S.daphnoides and S. incana x open pollinated hybrids was observed for the first time. Infection appeared in late spring (October-November), then declined almost dying out by mid-summer. Leaf tissues surrounding uredinia were distorted, bubbled and coloured yellow. The susceptibility of S. incana x open pollinated cultivars to this rust is listed in Table 2. In 1979 tree willows were infected by M. coleosporioides which entered New Zealand via trans-Tasman air currents from Australia (Latch, 1980). The host range and susceptibility of tree willows to M . coleosporioides is detailed in Table 3. Salix babylonica and most of its hybrids were highly susceptible whereas S. alba, S. nigm and S. mafsudana x S. alba were not infected. Salix matsudana cultivars were highly resistant and
many formed h e black spots and/or yellow flecks in response to infection. O n certain cultivars no uredinia but only h e telia formed. In 1981, light rust infection was observed on S. inferior, S. viminalis cv. Praecox, S. burjafica 'Germany', S. pontederana, S. nigricans fosferiana and S. osier Schmidt. Infection was not observed again until December 1985 when S. viminalis cultivars were infected and defoliated. Other, previously lightly infected cultivars attacked by M. epifea were also heavily infected. The host range and susceptibility of shrubby willow cultivars to this rust are presented in Table 4.
Hosf inoculation
Host inoculations (Table 5) established that the Melampsora pathogenic to S. daphnoidesls. incana x open pollinated hybrids was also pathogenic to S. caprea and S. hippophaefolia Kew. Melampsora epitea from S. caprea (pussy willow) was not pathogenic to S. viminalis cultivars, S. inferior or S. hippophaefolia Kew. The Melampsora from S. viminalis was pathogenic to S. inferior, S. viminalis cultivars, S. hippophaefolia (Kew) and also to several cultivars attacked by M. epifea (Table 5). Rust morphology
Uredinia of the Mehmpsora from S. daphnoides and S. incana x open pollinated cultivars were often surrounded by
A. G. Spiers and D. H. Hopcroft Table 5. Host range and susceptibility of willows to Melampsora epitea from S. caprea PG (Pussy willow), Melampsora from S virninalis and S. incana x daphnoides 'Tiritea' following inoculation and 15 d incubation at 20°
Accession number 215 233
1012 1211 1227 1244
Inoculum ex S. caprea
S. caprea 'PG' S. caprea 'N ' S. caprea x viminalis cv. Balana S. caprea x viminalis 'Srn' S. purpurea x viminalis cv. Populiferae S. caprea x viminalis cv. Hybrida S. cinerea 'W ' S. cinerea cv. Tricolor Mac S. hippophaefolia S. cinerea oleifolia S. aurita philicifolia ( = x Srnithiana) S. medemii S. mwcina S. hippophaefolia 'Kew' S. pontederana x gracilistyla S. caprea x viminalis 'SCCB' S. purpurea cv. Pyramidelis S. interior 'F' S. viminalis cv. Bowles hybrid S. purpurea x viminalis cv. Harrisons S. viminalis cv. Black Osier S. uiminalis cv. Praecox S. viminalis 'NCCB' S. viminalis 'M' S. uiminalis cv. Gigantea S. viminalis cv. Stone Osier S. incana x daphnoides cv. Tiritea S. glaucophyloides x viminalis cv. Gigantica S.incana x acutifolia S. incana x discolor
2 2 1
Odd pustule I 2 2 1 2 1 1 0 1 1
Odd pustule 0
Spots 0
Spots Spots 0
Spots 0
Spots 1 I I 2
' Rust 0, zero infection: 1, light infection 1-5 uredinia/m?; 2, heavy infection > 10 uredinia uredinia. a circle of orange-yellow coloured leaf tissue. Leaf surfaces were bubbled and distorted and uredinia were confluent and formed in a ring on both leaf surfaces. Single uredinia formed sub-epidermally and when fully expanded formed large cushions or bubbles on the leaf surface. In vertical section cells underlying the uredinium expanded and divided forming 4-5 layers at the base of the uredinium (Fig. 1).Single uredinia measured 1-2 x 0.5 mm. Uredinia were also formed on stems of young shoots. Uredinia tended to die out by mid summer leaving moribund white cushions on the leaf surface. Surrounding leaf tissue remained healthy and leaves did not fall prematurely. Uredinia of M. coleosporioides, M. epiitea and the Melampsora from 5. viminalis cultivars formed abaxially and were subepidermal (Figs 2-4). Single Uredinia were circular to oblong, and measured 0.125-0.30 mm diam. (Figs 5-7). Urediniospores Shape. Urediniospores of M. epitea, and the Melampsora spp. from 5. viminalis and 5. daphnoidesls. incana x open pollinated cultivars were globose to broadly ellipsoidal. Urediniospores of M. coleosporioides were obovate to pyriform (Figs 8-11). Spore walls of M . epitea, M. coleosporioides and Melampsora
Inoculum ex S. viminalis
Inoculum ex S. incana x daphnoides cv. Tiritea
Spots, necrotic fine spots; odd
1-2 scattered
from S. viminalis were thin (1.4 pm, mean; range 1.0-2.0 pm) whereas walls of Melampsora from 5. daphnoidesls. incana x open pollinated cultivars were thick (2.1 pm mean; range 1.4-3.0 m).Germpores were not evident in urediniospores of the Melampsora from S. viminalis. They were indistinct and partly visible in urediniospores of M. epitea and M . coleosporioides. Germpores were prominent in urediniospores of the Melampsora from 5. daphnoides/S. incana x open pollinated cultivars. Ornamentation. Urediniospores of M. coleosporioides were uniformly echinulate apart from a smooth apical spot. Urediniospores of the other Melampsora rusts were uniformly echinulate (Figs 8-11). Spines of all four Melampsora rusts were finely pointed, with similar profiles. Spines of M . coleosporioides were surrounded by basal annuli (Figs 12-15). Dimensions. The mean length and breadth and wall-thickness dimensions of urediniospores of M. epitea are listed in Table 1, the Melampsora from 5. incana x open pollinated cultivars (Table 2). M . coleosporioides (Table 3); and Melampsora from S. virninalis (Table 4). The coefficients of variation (Table 1-4) were generally less than 5% reflecting the uniform variability of mean length and breadth dimensions between
Studies of Melampsora rusts
Fig. 1. Vertical section through sub-epidermal uredinium of Melampsora ex S. incana on S. incana x daphnoides. Note the 4-5 layers of cells at the base of the uredinium. Also, the expansion and bubbling of the underlying leaf cells. Fig. 2. Vertical section through subepidermal uredinium of M . coleosporioides on S. babylonica. Note the elongate cells bearing the urediniospores and bubbling of the leaf surface. Fig. 3. Vertical section through subepidermal uredinium of Melampsora ex S. viminalis on S. interior. The uredinium is small and the leaf surface is undistorted. Fig. 4. Vertical section through subepidermal uredinium of M . epitea on S. caprea 'PG'. The uredinium is moribund as indicated by the vacuolate cells. hosts within species. The coefficients of variation for wallthickness varied from 5 % for M. 'viminalis' to 17.5 % for M. coleosporioides reflecting the variability of this morphological feature. The range and mean urediniospore length, breadth, wall thickness dimensions and ratios of the four 'species' are presented in Table 6. The mean length and breadth dimensions of the four 'species' were significantly (P < 0.05) different. Only urediniospore walls of the M e b m p sora from S. daphnoidesls. incana x open pollinated cultivars were significantly (P < 0.05) thicker than walls of the other 'species'. The L: B and L : W ratios of M. epifea and Melampsora from S. viminalis were not significantly different (P < 0.05). The L x B and L x B / W dimension of all Melampsora spp. were significantly different (P < 0.05). The mean length and breadth and wall thickness dimensions of the 16 herbarium specimens examined are listed in Table 7. These are further summarised and are presented along with ratios in Table 8. As with field collections, mean length and breadth dimensions of the four Melampsora spp. were significantly different (P < 0.05). The uredinioispore walls of Melampsora from S. daphnoidesls. incana x open pollinated cultivars were significantly thicker (P < 0.05) than other species. The L : B and L : W ratios of M . epitea and the Melampsora from S. viminalis were not significantly different ( P < 0.05). The L x B ratios of the four were significantly different (P < 0.05). The L x B / W ratios of M . epifea and the Melampsora from S. daphnoidesls. incana x open pollinated cultivars were not significantly different (P < 0.05).
Paraphyses. Paraphyses of the four Melampsom rusts were capitate and smooth-walled. Typically paraphyses of the
Melampsora from S. daphnoidesls. incana x open pollinated cultivars were thick-walled (3.0-5.0 pm), those of M . epitea were more variable, some being thin-walled (0.8-1.2 pm), others thick-walled (3.0-5.0 ym) and yet others apically thickened (4.0-5.0 pm). Paraphyses of Melampsom from S. viminalis were mostly thin-walled (1.2 pm and less) with some uniformly thick-walled (2-5-3.0 pm). Paraphyses of M. coleosporioides were uniformly thickened (1.5-2.0 pm), some with apices thickened (up to 5.0 ym). Paraphyses were difficult to measure in view of their variability, particularly the stalks which were easily broken. Accordingly, only the dimensions of the paraphyses head (length, breadth, wall thickness) were measured. These results are summarized in Table 9. Paraphyses heads of all four Melampsom rusts were significantly (P < 0.05) different in size.
Teliospores. Telia were not observed on Salix incana x open pollinated willow cultivars. Telia of the other rusts were single and scattered measuring 0.075-0.125 mm diam. to being confluent in large irreguIarly shaped clumps. Immature telia were amber-brown, turning dark-brown to black on maturity. Telia of M. coleosporioides were amphigenous although principally epiphyllous. They developed subcuticularly, intraepidermally and subepidermally. Telia of M . epifea were mainly hypophyllous and formed intra and subepidermally (Fig. 16).Telia of the Melampsora on S. viminalis cultivars were mainly epiphyllous and formed subepidemally (Fig. 17). Teliospores of M. coleosporioides were significantly (P < 0.05) longer and thinner than those of M. epitea and the Melampsora from S. viminalis, which were not significantly different. Teliospores of M. coleosporioides were rectangular to triangular,
A. G. Spiers and D. H. Hopcroft
1169
Figs 5-7. Fig. 5. Uredinium of Melampsora ex S. viminalis on the adaxial leaf surface of S. inferior. Fig. 6 . Uredinium of M . epifea on abaxial leaf surface of S. caprea 'PG'. Fig. 7. Uredinium of M. cokosporioides on abaxial leaf surface of S. babylonica.
38.6 5 5.1 pm in length (min. 20.0 pm, max. 50.0 pm), by 9.4 f 1.7 pm in breadth (min. 5.0 pm, max. 16.0 pm; Fig. 18). The side walls were 0.8-1.0 pm thick and were slightly thickened (1.5-2.0 pm) apically. Teliospores of M. epifea were also rectangular to triangular 29.8+4.2 pm in length (min. 16.0 pm, max. 40.0 pm) by 11.5 f 1.4 pm in breadth (min. 7.0 pm, max. 16.0 pm). Side walls were uniformly thin (0.5-0.8 pm). Teliospores of the Melampsora from S. viminalis were rectangular to triangular, 31.5 f 5.0 pm in length (min. 20.0 ym, max. 50.0 ym) by 12.4f2.6 pm in breadth (min. 7.0 pm, max. 20.0 pm). Teliospore walls were uniformly thin, measuring 0.5-0.8 pm. Metabasidia of M . coleosporioides were 40-60 pm long and composed of five cells, i.e. a basal cell 20 pm and four sterigmatal bearing cells 8.0-10.0 pm x 5.0-8.0 pm. Sterigmata measured 8.0-12.0-16.0 pm. Mature basidiospores were hyaline, smooth-waIled, apiculate and subgIobose, 6.3 k 0.5 x 5.2k0.5 pm (mean of 50 basidiospores). Metabasidia of M. epifea were 50-60.0 pm being composed of 4 sterigmatal bearing cells, 12.0-16.0 pm x 89-12.0 pm. Sterigmata measured 8.0-16.0-24.0 pm long. Mature basidiospores were hyaline, smooth-walled, apiculate
+
+
and subglobose, 10.5 1.2 x 9.5 1.1 pm (mean of 50 asidiospores). The metabasidium of the Melampsora from S. viminalis was 40.0-50.0 pm with four equally sized, sterigmatal bearing cells, 10.0-13.0 x 7.0-9.0 pm. Sterigmata measure 8.0-12.0 Urn long. Basidiospores were hyaline, smooth-walled, apiculate and subglobose, 9.0 0.6 x 8.7 & 0.9 pm (50 spores). Pycnia and aecia were formed only on Larix decidua plants inoculated with leaves bearing telia of M . epifea. Two pycnia were observed on needles of L. decidua inoculated with leaves bearing telia of M. coleosporioides. However, aecia failed to develop. No pycnia or aecia formed on L. decidua plants inoculated with leaves bearing telia of the Melampsora attacking 5. viminalis.
+
DISCUSSION Melampsora rusts of Salix are notoriously difficult to identify and knowledge of the aecial host is invaluable (Ziller, 1974; Pei ef al., 1993). Two taxonomic systems of willow rust taxonomy are recognized. One system represented by Klebahn (1904) and recognized by Sydow & Sydow (1915) and
Studies of Melampsora rusts
1170
Figs 8-11. Figs 8-10. Globose to broadly ellipsoidal, uniformly echinulate urediniospores of M. epifea (Fig.8), M. ex S. virninalis (Fig. 9 ) and M.ex S. incana x open poll. (Fig.10). Fig. 11. Obovate to pyrifonn, echinulate urediniospores of M. coleosporioides. Note the
partly bald apices on some urediniospores. Gauman (1959) treats rusts which are morphologically similar but with different alternate hosts as separate species. Gaumann (1959) recognized 17 species including a single autoecious species, M . amygdalinae Kleb. pathogenic to Salix friandra and S. pentandra. Within M. larici-epitea six fornzae speciales were recognized according to the Salix host. Pei et al. (1993, 1995) recently followed Gaumann (1959) when describing six Melampsora species from willows in England. An alternative taxonomic system lumped morphologically similar North American and European Melampsora species with different alternate hosts into a collective species, Melampsora epitea (Jorstad, 1940; Wilson & Henderson, 1966). Within M . epifea, two varieties, var. reficulatae (alternate host Saxifraga) and var. epitea were recognized. Within var. epitea were included forms alternating with Saxifraga (Formerly M. arctica); Euonymus (formerly M. euonymi-capraearum);Larix (formerly M . larici-epifea);Orchidaceae (formerly M . repentis); Ribes (formerly M. ribesii-purpureae); Tsuga (M. epifea f. sp. tsugae); Abies (M. abieti-capraearum); Larix (M. bigelowii = M . paradoxa) ( Ziller, 1959, 1974; Arthur, 1962; Wilson & Henderson). Species outside the M . epitea complex include: M . capmearum (alternate host Larix); M . larici-penfandrae (autoecious species); M . salicis-albae and M. allii-fragiles (alternate host Allium) (Wilson & Henderson, 1966).
Parmelee (1989) grouped Arctic willow rusts into M. epitea, hence followed Wilson & Henderson (1966). Ziller (1974) followed the conventional nomenclature of Arthur (1962), stating that critical revision of the group was necessary. Helfer (1992) also urged taxonomic revision of Melampsora on willows. He identified 14 Melampsora species affecting willows in Britain on the basis of spore and sorus characters confirming many species described by Klebahn (1904). Several other Melampsora species have been recorded in Asia including M . coleosporioides (Saccardo, 1905 ; Cummins, 1950; Cummins & Ling, 1950). Walker (1978) and Latch (1980) described M . coleosporioides in Australasia. Criteria used to delimit Melarnpsora species on willows have included the alternate host, apical thickening of aeciospore walls, size, thickening and location of teliospores, morphological features of urediniospores (dimensions, wallthickening, echinulation, germpores) and paraphyses (Arthur, 1962; Wilson & Henderson, 1966; Ziller 1974; Helfer 1992; Pei et. al., 1993, 1995). Morphological criteria used for delimiting Melampsora species on willows need to be identified. More detail is required in descriptions of urediniospores with regard to germpores, wall-thickening and dimensions. Mean length and breadth dimensions (of at least 50 spores) along with standard deviationlstandard error are more use than
A. G. Spiers and D. H. Hopcroft
1171
Figs 12-15. Finely pointed spines on urediniospores of M. epitea (Fig. 1 2 ) ; M. ex S. viminalis (Fig. 13);M. ex S. incana x open poll. (Fig. 14); and M. coleosporioides (Fig. 15). Note the annuli surrounding the spines of M. coleosporioides.
Table 6. Urediniospore dimensions and ratios of Melampsora species from field collections Urediniospore dimensions (pm) Length
M. coleosporioides M. epifea M. ex viminalis M. ex incann x open poll.
Breadth
Wall thickness
Ratios
range
mean
range
mean
range
mean
L:B
L/W
LxB
17.0-260 120-190 13.0-21.0 15.0-22.0
207A1 15.5D 16,OC 17.18
10.&160 10&16.0 11.5-16.0 12.0-19.0
13.7B 13.1D 13.4C 15.6A
1.0-2.0 1-&20 0q2.0 1-4-3.0
1.48 1.4B 1.48 2.1A
1.51A 1.18B 1.198 1.09C
14.80A 11.07B 11.42B 8.14C
283.6A 203.00 214.4C 266.78
Lx B -
W 202-6A 145.0C 153.1B 1270D
Means not followed by the same letter differ significantly (Pi 0.05) according to Duncan's multiple range test.
vague figures usually quoted, i.e. 14-21 x 13-16 ym. The same applies to dimensions of paraphyses, telia and aeciospores. Host range studies are compounded by the difficult taxonomy of Salix species particularly between the Northern and Southern Hemisphere countries. In the present study the influence of the host on urediniospore dimensions was small ( < 4%), and insufficient to obscure rust species differences. There is a need to determine the extent of variability expressed by various morphological features of Melampsora
willow rusts and to decide which features can best be used to differentiate species, that is, the steps 'analysis' and 'synthesis' used by Snyder & Hansen (1954) in their revision of the genus Fusarium.
The present study has shown that morphological features of urediniospores, namely echinulation, length and breadth dimensions, wall thickness, the presence of germpores, and L x B ratios were useful criteria for describing and delimiting species. Urediniospores of M. coleosporioides and the M e l a m -
Studies of Mebmpsora rusts
1172
Table 7. Herbarium collections of willows infected with Mebmpsora spp. examined and dimensions of urediniospores Urediniospore dimensions (pm)
M . c~leos~orioides M . c~leos~orioides M. coleosporioides M . coleosporioides M . ex S.incana x open M . ex S. incana x open M . ex S. incana x open M . ex S. incana x open M . ex S. viminalis M . ex S. viminalis M . ex S. viminalis M . ex S. viminalis M . epiten M. epitea M. epitea M. epitea
poll. poll. poll. poll.
Collection date
Host
Locality
S. babylonica S. babylonica S.babylonica S. babylonica
Whangarei Palmerston North Wanganui Palmerston North Palmerston North Palmerston North Palmerston North Palmerston North Palmerston North Christchurch Palmerston North Palmerston North Palmerston North Palmerston North Turangi Waiuku
N Z 1244 N Z 1009 N Z 1227 NZ 1226 S. interior S. viminalis S.viminalis ' M ' S. viminalis cv. Bowles hybrid S. hippophaefolia S.hippophaefolia 5. cinerea S. cinerea
Length
Breadth
Wall thickness
Means not followed by the same letter differ significantly ( P < 0.05) according to Duncan's multiple range test.
Table 8. Urediniospore dimensions and ratios of Melampsora species from herbarium specimens Urediniospore dimensions (pm)
M . coleo~~orioides M . epitea M. ex S. vimirraiis M. ex S.incana x open poll.
Ratios
Length
Breadth
Wall thickness
21.1A1 15.1D 163C 1758
13.3C 12.9D 14.28 15.9A
1.48 1.48 1.48 2.OA
L:B
L/w
Lx B
LxB W
Means not followed by the same letter differ significantly ( P < 0.05) according to Duncan's multiple range test
Table 9. Urediniospore dimensions and ratios of Melampsora species from herbarium specimens Paraphyses dimensions (pm) Length
M. coleosporioides M . epitea M. ex S. viminalis M . ex S.incana x open poll.
Breadth
Wall thickness
Range
Mean
Range
Mean
Range
Mean
LxB
160-25.0 16.0-34.0 160-300 16.0-35.0
18.1D1 22.6C 23.28 24.3A
12.0-18.0 12.0-24.0 12.0-24.0 12.0-24.0
15.7D 16.6C 18.6A 17.28
08-3.0 08-64 084.4 08-8.0
1.9D 3.08 2.5C 4.8A
284.2D 375.2C 431.5A 41798
Means not followed by the same letter differ significantly (P < 0.05) according to Duncan's multiple range test.
psora from S. daphnoides and S. incana x open pollinated cultivars were morphologically distinct, i.e. the elongate, thinwalled urediniospores of the former compared with the thickwalled, germpore - studded urediniospores of the latter. Urediniospores of Melampsora epitea and the Melarnpsora from S. viminalis were morphologically similar. However with familiarity, the significantly (P < 0.005) smaller, less regular urediniospores of M. epitea, with germpores, were easily distinguished from the regular, germpore free, uniformly thinwalled urediniospores of the Melarnpsora from S. viminalis. Paraphyses were of limited use for species identification. Nevertheless, the small, thin-walled paraphyses of M . coleo-
sporioides were significantly smaller than those of the other Melampsora species. The large paraphyses of the Melampsora attacking S. daphnoides and S. incana x open pollinated cultivars were generally more thick-walled than those of the other Melampsora 'species', although there was much overlap. However, in view of their variability, paraphyses should only be considered as secondary characters for use in species identification. Location of teliospores, both with regard to leaf surface and leaf tissue (subcuticular, intraepidermal, subepidermal) was not a stable morphological feature and therefore not suitable for species identification. Telia of M . epitea mostly formed on
A. G. Spiers and D. H. Hopcroft
Figs 16-18. Fig. 16.Subepidermal, hypophyllous telia of M. epitea. Fig. 17. Subepidermal, amphigenous telia of M ex S. viminniis. Fig. 18. Intraepidermal, epiphyllous telia of M. coleosporioides.
the underside of leaves, whereas telia of the Melampsora attacking S. uiminalis were epiphyllous. Telia of M. coleosporioides were amphigenous. Wilson & Henderson (1966) reported that telia of M. capraearum and M. ribesii-viminalis were epiphyllous, whereas, although telia of M . epifea were mostly hypophyllous, they could also be epiphyllous. Within leaf tissues, telia of M . coleosporioides were sub-cuticular, intraepidermal and subepidermal whereas those of M. epitea and the Melampsora attacking S. viminalis were intraepidermal and subepidermal. No doubt subcuticular telia were also formed. Subcuticular telia were considered to be diagnostic features of M. capraearum and M. ribesii-viminalis by Wilson & Henderson (1966). The thickening (or lack of it) of teliospore walls can be a stable diagnostic feature. In the present study, telia walls were uniformly thin and lacked germination pores. The thickened apical, porate telial wall of M. capraearum was considered to be diagnostic of that species by Wilson & Henderson (1966). Teliospore length and breadth dimensions have not been used to delimit species (Arthur 1962; Wilson & Henderson, 1966), possibly because they are difficult to separate and measure accurately. Teliospores of M. coleosporioides were significantly (P < 0.05) taller and thinner than those of M . epifea and the Melampsora attacking S. uiminalis, which were not significantly different ( P < 0.05).
Morphological features of metabasidia and basidiospores have not been used to characterize Melampsora willow rusts. Ziller (1959) described basidia of M . epifea f. sp. tsugae. In the present study, the metabasidium and basidiospores of M. coleosporioides, M . epitea and the Melampsora attacking S. uiminalis were all significantly different (P > 0.05) in size, particularly the diameter of the metabasidium and the basidiospores, those of M. epifea being almost twice those of M . coleosporioides. Clearly, morphological features and dimensions of metabasidium and basidiospores should be considered, where possible in the description of Melampsora species. Teliospores were not observed on S. daphnoides and S. incana x open pollinated cultivars, yet this rust has overwintered successfully for several years. M . epitea, the Melampsora attacking S. virninalis and M. coleosporioides can also overwinter in the absence of alternate hosts. In the case of the Mehmpsora on S. uiminalis, new uredinia are observed in spring, on the first-formed leaves, I month following budburst. Notwithstanding, infection has been observed to overwinter only on certain cultivars of S. viminalis and never on S. interior. These rusts possibly overwinter as urediniospores or as uredinial mycelium as commonly reported for Melampsora willow rusts (Savile, 1953; Wilson & Henderson, 1966; Ziller, 1974); Parmelee (1989) and Pei ef al., (1995) described a stem-infecting form of M. epitea var. epifea f. sp. hrici-epitea typica attacking S. viminalis which overwintered as urediniospores in buds and stems. M. coleosporioides also overwinters as uredinia on persistent leaves retained on trees. Although telia of this rust germinate profusely in spring they appear to play no role in the life cycle. The alternate host of M. coleosporioides has not been determined (Spiers & Hopcroft, 1988). Melampsora epifea can readily alternate through larch wherever willows and larches are planted together (Spiers & Hopcroft, 1985). In the present study, M. coleosporioides was morphologically distinct from the other Melampsora species and was the only species pathogenic to tree willows. This rust was identified as M . coleosporioides by Walker (1978) and Latch (1980). The rust attacking shrub willows has previously been identified as M. epitea by Dingley (1977). Because the aecial stage of this rust occurs on Larix (Spiers & Hopcroft, 1985), the rust could be classified as M. epitea var. epitea (Wilson & Henderson, 1966), or M. larici-epitea f. sp. fypica (Gaumann, 1959). If the broad species concept of Wilson & Henderson (1966) were followed the Melampsora attacking S. viminalis could also be classified as M. epitea var. epifea (M. epifea sensu J0rstad (1940)). The alternate host of this rust needs to be determined. On the other hand, if Gaumann (1959) was followed the rust would be classified as M. larici-epitea f. sp. fypica. Neither classification is satisfactory since the Melampsora attacking S. viminalis is not wholly morphologically and physiologically similar to M. epitea var. epitea. For example, it has significantly larger (P < 0.05) urediniospores and paraphyses (with thinner walls), epiphyllous telia (cf. hypophyllous) and does not have Larix decidm as an alternate host. Although it shares some Salix hosts with M . epitea var. epifea it tends to be pathogenic to S. viminalis and its hybrids. This rust does not appear to be M. ribesii-viminalis Kleb. because its urediniospores are smaller with thinner walls. Both have thickened paraphyses and
Studies of Melampsora rusts epiphyllous teliospores which are subcuticular in M . ribesiiviminalis (sub-epidermal for New Zealand rust) (Wilson & Henderson, 1966). A race of M . epifea with sub-epidermal teliospores and thickened paraphyses has been recorded on S. virninalis in Europe (Wilson & Henderson, 1966). The Melampsora from S. daphnoides and S. incana x open pollinated cultivars does not fit into the M. epifea complex in view of its thickened urediniospore walls with prominent germspores and large, thick-walled paraphyses. This rust with its large uredinal pustules and distorted leaf surfaces does not fit published descriptions (Jorstad, 1940; Gaumann 1959; Arthur, 1962; Wilson & Henderson, 1966; Ziller, 1974; Parmelee, 1989; Helfer, 1992). In dimensions of urediniospores, wall thickening and germpores it broadly resembles M. capraearum Thiim (Wilson & Henderson, 1966), or M. laricicapraearum Kleb. (Gaumann, 1959). The absence of teliospores hinders confirmation of this. M . larici-epifea f. sp. laricidaphnoides Kleb. has similar urediniospore morphology and willow host range but has telia and alternates via larch (Gaumann, 1959). Clearly, the identity of this rust remains uncertain. It is unusual in that it causes leaf distortion, possibly though the production of growth substances, lacks telia, overwinters in buds and tends to die out by mid summer. How did these Melampsora rusts enter New Zealand? Melarnpsom epifea was first recorded in New Zealand by Dingley (1977) although the rust had been in the country at least 10 years previously (C. W. S. Van Kraayenoord, pers. comm.) Walker (1978) reported that M . epitea had been known in New South Wales and Victoria since 1972. It is unknown whether the rust entered New Zealand from Australia or whether it was introduced independently into both countries. Similarly, it is unknown how the rust attacking S. daphnoides entered New Zealand in 1978. Melampsora coleosporioides entered Australia illegally and was first recorded in April 1978 (Walker, 1978). It crossed the Tasman via wind currents and was recorded in Whangarei, New Zealand in November 1978 (Latch, 1980). The Melampsom attacking S. virninalis appeared briefly on several cultivars in 1981, died out and then reappeared on S. viminalis throughout New Zealand in December 1985. The sudden appearance of this rust throughout New Zealand coincided with that of Marssonina casfagnei which was also recorded on silver poplar throughout New Zealand for the first time in December 1985 (Spiers, 1988). Previously M. casfagnei had entered Australia illegally and was first reported there in February 1984 (J. C. Walker, pers. comm.). The S. viminalis rust is probably also of Australian origin. One possible explanation for the blanket distribution of this Melampsora rust and Marssonina casfagnei throughout New Zealand could be that the fungi were transported via trans-Tasman wind currents which entered New Zealand at the base of and/or top of the country and moved up or down the length of New Zealand depositing conidia and uredinospores. Alternatively several independent trajectories may have been involved. The meteorological aspects of trans-Tasman dispersal have been reviewed by Tomlinson (1973) and Close et al., (1978). This study has shown that the current taxonomy of Melampsora species is inadequate. This problem is overcome at present by lumping difficult to identify species into
1174 complexes like M. larici-epitea (Gaumann, 1957), or M. epifea (Wilson & Henderson, 1966). As suggested by Helfer (1992), a taxonomic revision of Melarnpsora is advocated. We thank Raymond Bennett for the preparation of the electron micrographs.
REFERENCES Ager, A., Ronnberg-Wastljung, A., Thorsen, J. & Siren, G. (1986). Genetic improvement of willows for energy forestry in Sweden. Report No. 43, Swedish University of Agricultural Sciences, Uppsala, Sweden. Arthur, J. C. (1962). hfanual of Rusts rn United States and Cai~ada.Hafner Publ. Co.: New York. Close, R. C., Moar, N. 7.. Tomlinson, A. I. & Lowe, A. D. (1978). Aerial dispersal of biological material from Australia to New Zealand. International Journal of Biometeorology 22. 1-19, Cummins, G. B. (1950). Uredinales of continental China collected by S. Y. Cheo. 1. Mycologia 42. 779-797. Cummins, G. B. & Ling, L. (1950). An index of the plant rusts recorded for continental China and Manchuria. Plant Disease Reporter Supplement 196, 52&556. Dawson, W. M. (1988). Production of biomass from short-rotation coppice willow in Northern Ireland 1974-1987. In Proceedings of the IEA Task I1 Meeting and Workshops on Cell Culture and Coppicing, Oulu, Finland, 24-29 Augwt 1987 (ed. Ari. Ferm). Dingley, J. M. (1977). Additions to the rust fungi of New Zealand. VI. New Zealand Journal of Botany 15, 29-37. Freund, F. J. & Littell, R. C. (1981). SAS for Linear Models. A Guide to tlte Anova and G L M Procedures. SAS Institute Inc.: Cary, North Carolina. Gaumann, E. (1959). Die Rost~ilzeMitteleuropas. Beitrage zur Kryptogamenflora der Schweiz 12. Buchdruckerei Buchler & Co.: Bern. Helfer, S. (1992). The rust diseases of willows in Britain. Proceedings of the Royal Society of Edinburgh 988, 119-134. Jarstad, I. (1940). Uredinales of Northern Norway. Skr$ter Norske Videnskaps Akad. Mat. Nat. KI. 6, 1-45. Klebahn, H. (1904). Die wirtswechseladen Rostpike. Gebriider Borntraeger: Berlin. Latch, B. J. (1980). Weeping willow rust in New Zealand. New ZealandJournal of Agrictcltural Research 23, 535-538. McCracken, A. R. & Dawson, M. (1992). Clonal response in Salix to Melampsora rusts in short rotation coppice plantations. European Journal of Forest Pathology 22, 19-28. Pamelee, J. A. (1989). The rusts (Uredinales) of arctic Canada. Canadian Journal of Botany 67,3315-3365. Pei. M. H., Royle, D. J. & Hunter, T. (1993). Identity and host alternation of some willow rusts (Melampsora spp.) in England. Mycological Research 97, 845-851. Pei, M. H., Royle, D. J. & Hunter, T. (1995). A comparative study of stem- and leaf-infecting forms of Melampsora rust on Salix viminalis in the U.K. Mycological Research 99.357-363. Ronnberg-Wastljung, A. G. & Gunnerbeck, E. (1985). Infection patterns and host differences in Melampsora attacks on Salix ~lantations.Report 38.
Energy Forest project, Department of Ecology and Environmental Research, Swedish University of Agricultural Sciences, Llppsala. Saccardo, P. A. (1905). Sylloge fungorum omnium hucwque cognitorum 17, 265-266. Pavia, Italy. Savile, D. B. 0. (1953). Short season adaptations in the rust fungi. Mycologia 45, 75-87. Schreiner, E. J. (1959). Rating poplars for Melampsora leaf rust infection. Foresty Research Notes, Northeast Experimental Station 90. Snyder, W. C. & Hansen, H. N. (1954). Variation and speciation in the genus Fusarium. Annals of the New York Academy of Sciences 60, 16-23. Spiers, A. G. (1988). Studies of Marssonina castagnei in Australasia. European Journal of Foresf Patkology 18, 65-76.
A. G. Spiers and D. H. Hopcroft Spiers, A. G. & Hathaway, R. L. (1987). Melampsora rusts of Salix in New Zealand. International Energy Agency, Task I1 - Biomass growth and production technology meeting, Oulu, Finland. Uppsala, Sweden. Spiers, A. G. & Hopcroft, D. H. (1985). Ultrastructural studies of the spermat~aland aecial stages of Melampsora larici-populina and Melampsora epitea on Larix decidua. New Zealand Journal of Botany 23, 101-116. Spiers, A. G. & Hopcroft, D. H. (1988). Ultrastructural studies of the telial, basidial, and spermatial stages of the willow rust fungus Melampsora coleosponoides in New Zealand. New ZealandJournal of Botany 26, 423-430. Sydow, P. & Sydow, H. (1915). Monographia Uredinearum 3. Leipzig. Tomlinson, A. I. (1973). Meteorological aspects of trans-Tasman insect dispersal. New Zealand Entomologist 5, 253-268. Van Kraayenoord, C. W. S. (1975). Willows. New Zealand Nature Heritage 7 (98), 2730-2737. Van Kraayenoord, C. W. S., Laundon, G. F. & Spiers, A. G. (1974). Poplar rusts invade New Zealand. Plant Disease Reporter 58, 423-427.
(Accepted 76 February 1996)
1175 Van der Meiden, H. A. (1961). De betekenis van enkek bladzickten van der populier in de populieren-feelt. (The importance of some poplar leaf diseases in poplar culture) Bericht nr. 43. Bosbouwproefstation: Wageningen. Verwijst, T. (1990). Clonal differences in the structure of a mixed stand of Salix viminalis in response to Melampsora and frost. Canadian Journal of Forest Research 20, 602-605. Walker, J. C. (1978). Rust on weeping willow in Australia. Australasian Plant Pathology 7,34. Wilson, M. & Henderson, D. M. (1966). British Rust Fungi. Cambridge University Press: Cambridge U.K. Ziller, W. G. (1959). Studies of Western tree rusts. V. The rusts of Hemlock and Fir caused by Melampsora epitea. Canadian Journal of Botany 37, 109-119. Ziller, W. G. (1974). The tree rusts of Western Canada. Canadian Forestry Service Publication No. 1329.
Mycol. Res. 100 (10): 1163-1175 (1996) Printed in Great Britain
Morphological and host range studies of Melampsora rusts attacking Salix species in New Zealand
A. G. SPIERS A N D D . H. HOPCROFT The Horticulfure and Food Research Institute of New Zealand Lfd, Palmersfon North, New Zealand
Comparative studies of Melampsora rusts pathogenic to willows in New Zealand showed that recognition of two named species was possible. These were M . coleosporioides, attacking tree willows, particularly S. babylonica and M . epifea var. epitea (according to Wilson & Henderson), and M . brici-epifea f. sp. larici-epifea typica (according to Gaumann), which was pathogenic to S. cinereal5 caprea with Larix as an alternate host. Two other Melampsora msts pathogenic to S. viminalis and S. daphnoidesls. incana x open pollinated hybrids respectively were not identifiable to species level from published descriptions. Useful taxonomic criteria included morphological features of urediniospores (shape, dimensions, ratios, wall-thickness, germpores), dimensions of paraphyses heads, teliospores, metabasidia and basidiospores. A plea is made for revision of willow rust taxonomy and for more detailed rust descriptions.
Willows have been extensively planted throughout New Zealand since their introduction in the 1840s. They have been utilized for amenity plantings, i.e. S. babylonica (weeping willow), S. alba var. vitellina pendula (golden weeping), 5. x sepulcralis ( S . babylonica x S. alba), S. matsudana (Peking willow), S. mafsudana cv. Tortuosa (corkscrew willow), S. alba var. vitellina (golden willow), S. incana, S. purpurea, S. viminalis, S. matsudana x S. alba (hybrid willows); for soil conservation, i.e. S. mafsudana, S. mafsudana x S. alba and horticultural shelter, i.e. S. matsudana, S. matsudana x S. alba and S. x caprea (pussy willow). The osier willows, S. purpurea, S. triandra and S. viminalis have also been utilized to a limited extent for basketware (Van Kraayenoord, 1975). Leaf rust caused by Melampsora species is the most important growth-limiting disease afflicting willows in New Zealand. Extensive monoclonal planting of a few select cultivars is being avoided by planting cultivar mixture and having a number of genetically diverse cultivars available. The vulnerability of monoclonal plantings to rust in New Zealand was clearly demonstrated by the impact of Melampsora laricipopulina and M . medusae on poplar plantations (Van Kraayenoord et al., 1974). In Canada, England and West European countries, willows (S. viminalis clones and other Salix spp.) have been grown for renewable energy (Ager ef al., 1986; Venvijst, 1990; McCracken & Dawson, 1992). Foliar rust caused by Melampsora species has been a major problem, causing premature defoliation and markedly reduced yields (McCracken & Dawson, 1992) which has highlighted the problems of willow rust identification (Dawson, 1988; Ronnberg-Wastljung & Gunnerbeck, 1985; Pei et. al., 1993, 1995). There is much confusion regarding the identity of
Melampsora species attacking willows in New Zealand. There appear to be four morphologically distinct rusts. Two of these, M. epifea Thiim. and M . coleosporioides Dietel, have been briefly described by Dingley (1977) and Latch (1980) respectively, while the two other rusts attacking cultivars of S. viminalis and S. daphnoides have been briefly described by Spiers & Hathaway (1987). In this paper the morphology and host range of Melampsora species attacking willows in New Zealand are described.
MATERIALS A N D M E T H O D S Host range and susceptibility A willow cultivar collection held as stools (five stools per cultivar) at Aokautere, Palmerston North was rated for Melampsora infection during March and April each year. Monitoring commenced in 1977 and is continuing. Rust severity was assessed using the Schreiner-van der Meiden rating system (Schreiner, 1959; van der Meiden, 1961). Host records were confirmed by microscopic measurement of uredinial features.
Host inoculation Inoculations of detached leaves (Table 5) were conducted to clarify the host ranges of M. epitea and the rusts attacking S. viminalis and S. daphnoidesl.5. incana x open pollinated cultivars. Inoculations were made in December when fresh urediniospores and rust-free leaves were available. Leaf discs (20 mm diam.) and small leaves were inoculated abaxially by brushing on urediniospores. Discs and leaves
Studies of Melampsora rusts
1164
Table 1. Host range, susceptibility and uredinial dimensions of Melampsora epitea from shrub willows Urediniospore dimensions ( p m ) Rust rating (March-April)
Accession number
S. hippophaefolia S. cinerea cv. Tricolor Mac 5. incana discolor S. aurita var. philicifolia S. cinerea oleifolia S. cinerea 'M' S. cinerea 'W' S. silesiaca ( x calodendron) 5. caprea x uiminalis cv. Balana 5. caprea x uiminalis 'SM' S. caprea x uiminalis 'SCCB' 680 S. dichroa (aurita x purpurea) 367 S. seringeana 667 S. purpurea cv. Pyramidalis 309 S. caprea F (5. x reichardtii) S. purpurea x uiminalis Populifera 301 332 5. pontederana x gracilisytla 215 S. caprea 'PG' (discolor B) 229 5. medemi (aegyptiaca) 306 S. caprea x uiminalis 'Hybrida' 714 S. mwcina 237 5. pontederana 346 S. pip0 12 347 S. pip0 13 S. osier cv. Schmidt 628 Mean urediniospore dimensions Coefficient of variation (CV%)%
Length
Breadth
14.9 15.2 15.6 16.7 15.9 16.3 15.5 14.9 14.7 15.8 14.9 15.5 15.3 15.3 15.6 15.6 16.3 16.9
13.0 13.0 13.1 12.5 14.4 13.6 12.6 12.6 12.7 14.3 13.0 13.3 13.1 126 13.6 13.4 13.7 13.5
Wall thickness
224 324
Not Not Not Not
measured measured measured measured
15.5 3.8
13'1 4.2
Rust rating = Schreiner-van der Meiden rating system. 2
C V % coefficient of variation = standard deviation (100). mean
Table 2. Host range, susceptibility and urediniospore dimensions of Melarnpsora epitea from S. incana cultivars Urediniospore dimensions ( p m ) Accession number
Rust rating (March-April)
Length
Breadth
Wall thickness
S. daphnoides var. acutifolia S. daphnoides ' G ' S. incana x open pollinated S. incana x open pollinated S. incana x open pollinated S. incana x open pollinated S. incana x open pollinated S. incana x acutifolia S. incana x acutifolia S. incana x acutifolia 5. incana x acutifolia 5. incana x acufifolia Mean urediniospore dimensions Coefficient of variation (CV%)2 331 213 NZ1005 NZ1006 NZ1008 NZ1009 NZlOl2 NZ1228 NZ1230 NZ1237 NZ1226 NZ1227
Rust rating = Schreiner-van der Meiden rating system. 2
CV%
of variation = standard
mean
(100)
were incubated on moistened filter paper under natural light at 20 OC. Following 7, 10 and 15 d incubations, discs were examined for erumpment uredinia. Identification of Melampsora rusts was confirmed by microscopic examination of urediniospores.
Rust morphology
Rust samples were collected from willows growing at Aokautere during March (Tables 1-4). For each collection, the host species and gross features of uredinia and telia (when
A. G. Spiers and D. H. Hopcroft
1165
Table 3. Host range, susceptibility and urediniospore dimensions of Melampsora coleosporioides from shrub willows Urediniospore dimensions (pm) Accession number
Rust rating (March-April)
S. babylonica x humboldtiana '160-16' S. argentinensis cv. Mestizo Pereyra S. babylonica S. babylonica var. annularis 5.babylonica x fragilis cv. Blanda S. babylonica x fragilis 5.babylonica x fragilis S. elegantissima (babylonica x fragilis) S. fragilis hybrid cv. Benmore S. pendulina (babylonica xfragilis) S. chilensis S. matsudana tortuosa 'NG' 5.pentandra 'G' S. penetandra cv. Dark French S. pentandra cv. Patent Lumley S. matsudana 'LG105' S. matsudana pendula 'CHI' S. matsudana ' CH2 ' S. matsudana war. turiuosa cv. Northland A S. matsudana var. tortuosa cv. Northland B S. matsudana cv. Nelson purple S. meyeriana S. salamonii S. sepulchralis (babylonica x alba) S. matsudana var. tortuosa S. matsudana 'CH4' S. babylonica x alba '131-25 ' S. babylonica x alba '131-27' S. matsudana 'YN 102' S. matsudana 'YN 207' S. matsudana 'CH 3B' S. matsudana cv. Nelson green S. coerulea 'M' S. matsudana 'CH3A' S. himalayas s.fragiiis '9/17' S. fragilis Illectic V 10/30 25 9 S. elegantissima (babylonica x fragilis) Mean urediniospore dimensions 661 658 292 321 217 277 311
(CV%)4
Length
Breadth
19.7 21.4 205 21.5 20.8 21.4 19.6 20.9 19.6 20.3 207 20.8 20.0 21.0 20.9 21.0 20.9 21.5 206 21.4 202
13.7 13.3 13.3 13.9 12.8 13.2 13.8 13.4 14.3 13.2 13.5 14.3 13.9 13.3 13.7 13.6 14.1 13.9 14.5 14.7 13.7
Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not 20.7 2.9
Wall thickness
formed formed formed formed formed formed formed formed formed formed formed formed formed formed formed formed formed 13.7 3.6
Rust rating = Schreiner-van der Meiden rating system. S = fine yellow or black spots. T = fine telia formed. 4
CV% coefficient of variation
= standard
mean
(100).
formed) were recorded. Urediniospores were mounted in lactophenol and stained with 0.5% acid fuchsin. Sixty uredinospores per collection were measured using a Leitz micrometer (under interference contrast) at 5 0 0 ~ magnification. Dimensions recorded were urediniospore length, breadth and wall thickness. From these, ratios of length to breadth (L:B); length to wall thickness (L:W); length by breadth (L x B) and length by breadth over wall thickness (L x B/W) were calculated. Urediniospore dimensions and ratios were analysed by the SAS procedure GLM (Freund & Littell, 1981). The length and breadth dimensions and wall thickness of paraphyses heads were measured. Also, echinulation and presence of germpores in urediniospores was recorded. Sixteen
collections of Melampsora (four of each possible species) from the Aokasutere herbarium were also examined. Urediniospore dimensions and ratios were measured and calculated as detailed above for field collections. To determine the morphology and location of uredinia and telia, small leaf sections (1x 5 mm) were prepared for transmission electron microscopy as previously described (Spiers & Hopcroft, 1985). Urediniospore morphology was examined by scanning electron microscopy (Spiers& Hopcroft, 1985). During late autumn (April-May) leaves bearing teliospores of M. epifea, M . coleosporioides and the Melampsora infecting 5. viminalis were collected and stored dry, in the dark at zO,for 5 months. To induce telial germination the leaves were
Studies of Melampsora rusts
1166
Table 4. Host range, susceptibility and urediniospore dimensions of Melampsora sp. from S. viminalis cultivars Urediniospore dimensions (vm) Accession number
Rust rating (March-April)
Length
Breadth
Wall thickness
S. purpurea x viminalis cv. Populifera S.inferior 'F ' S. purpurea x viminalis cv. Harrison S. viminalis 'NCCB' S.viminalis cv. Praecox S. viminalis cv. Black Osier S. viminalis cv. Stone Osier S.viminalis 'M' S. purpurea x viminalis sessifolia S. purpurea pyramidalis S. viminalis cv. Gigantea S. viminalis cv. Bowles hybrid S. pontederana x gracilis S. incana x discolor S. caprea x viminalis 'SM' S.caprea ' N ' S. pentederana S. hippophuefolia 'Kew' S. hippophaefolia (ex mollisima var. trevirani) 204 S. aurita var. philicifolia S. x reichardtii (5.caprea F) 215 234 S. nigricans fosteriana 668 5. burjatica 'Germany' 634 S.walsfeiniana 609 S. purpurea cv. Green Dicks Mean urediniospore dimensions (CV%)2
375' 3 75 375 3 75 3 75 3 75 250 250 250 125 125 125 50-75 25 25 10-25 5 5 5
15.6 15.9 15.9 15.4 16.8 14.9 15.5 174 16.8 18.5 15.4 18.5 15.8 16.1 15.7 14.7 15.5 15'4 15.1
13.4 13-4 13.7 13.3 12.9 13.2 13.3 13.5 12.9 13.6 12.9 13.9 13.5 13.8 12.8 12.8 13.3 13.4 13.5
1.6 1-4 1-4 1.4 1.4 1.4 1.3 1.5 1.5 1.5 1.3 1.5 1.4 1.4 1.4 1-4 1.5 1.5 1.4
5 5 4 4
16.4 15.8 16.0 15.6 15.6 15.3 16.0 6.0
13.6 13.6 13.4 13.4 13.6 13.6 13.4 3.0
1.5 1.4 1.4 1.5 1-5 1-4 1.4 5.0
301 687 607 245 272 3 75 379 275 305 667 220 312 332 1244 303 233 237 383 224
4
2
See footnotes, Table I.
rehydrated and placed outside among year-old plants of Larix decidua, in separate enclosures. Dimensions of metabasidia and basidiospores were measured.
RESULTS Hosf range and susceptibility
A single Melampsom species was observed on Salix when observations commenced in 1977. This species was identified as Melampsora epitea (Dingley, 1977) and was found to have Larix decidua and L. leptolepis as alternate hosts (Spiers & Hopcroft, 1985).Table 1lists the host range and susceptibility of shrub willows to M. epitea. During 1978, rust infection of S.daphnoides and S. incana x open pollinated hybrids was observed for the first time. Infection appeared in late spring (October-November), then declined almost dying out by mid-summer. Leaf tissues surrounding uredinia were distorted, bubbled and coloured yellow. The susceptibility of S. incana x open pollinated cultivars to this rust is listed in Table 2. In 1979 tree willows were infected by M. coleosporioides which entered New Zealand via trans-Tasman air currents from Australia (Latch, 1980). The host range and susceptibility of tree willows to M . coleosporioides is detailed in Table 3. Salix babylonica and most of its hybrids were highly susceptible whereas S. alba, S. nigm and S. mafsudana x S. alba were not infected. Salix matsudana cultivars were highly resistant and
many formed h e black spots and/or yellow flecks in response to infection. O n certain cultivars no uredinia but only h e telia formed. In 1981, light rust infection was observed on S. inferior, S. viminalis cv. Praecox, S. burjafica 'Germany', S. pontederana, S. nigricans fosferiana and S. osier Schmidt. Infection was not observed again until December 1985 when S. viminalis cultivars were infected and defoliated. Other, previously lightly infected cultivars attacked by M. epifea were also heavily infected. The host range and susceptibility of shrubby willow cultivars to this rust are presented in Table 4.
Hosf inoculation
Host inoculations (Table 5) established that the Melampsora pathogenic to S. daphnoidesls. incana x open pollinated hybrids was also pathogenic to S. caprea and S. hippophaefolia Kew. Melampsora epitea from S. caprea (pussy willow) was not pathogenic to S. viminalis cultivars, S. inferior or S. hippophaefolia Kew. The Melampsora from S. viminalis was pathogenic to S. inferior, S. viminalis cultivars, S. hippophaefolia (Kew) and also to several cultivars attacked by M. epifea (Table 5). Rust morphology
Uredinia of the Mehmpsora from S. daphnoides and S. incana x open pollinated cultivars were often surrounded by
A. G. Spiers and D. H. Hopcroft Table 5. Host range and susceptibility of willows to Melampsora epitea from S. caprea PG (Pussy willow), Melampsora from S virninalis and S. incana x daphnoides 'Tiritea' following inoculation and 15 d incubation at 20°
Accession number 215 233
1012 1211 1227 1244
Inoculum ex S. caprea
S. caprea 'PG' S. caprea 'N ' S. caprea x viminalis cv. Balana S. caprea x viminalis 'Srn' S. purpurea x viminalis cv. Populiferae S. caprea x viminalis cv. Hybrida S. cinerea 'W ' S. cinerea cv. Tricolor Mac S. hippophaefolia S. cinerea oleifolia S. aurita philicifolia ( = x Srnithiana) S. medemii S. mwcina S. hippophaefolia 'Kew' S. pontederana x gracilistyla S. caprea x viminalis 'SCCB' S. purpurea cv. Pyramidelis S. interior 'F' S. viminalis cv. Bowles hybrid S. purpurea x viminalis cv. Harrisons S. viminalis cv. Black Osier S. uiminalis cv. Praecox S. viminalis 'NCCB' S. viminalis 'M' S. uiminalis cv. Gigantea S. viminalis cv. Stone Osier S. incana x daphnoides cv. Tiritea S. glaucophyloides x viminalis cv. Gigantica S.incana x acutifolia S. incana x discolor
2 2 1
Odd pustule I 2 2 1 2 1 1 0 1 1
Odd pustule 0
Spots 0
Spots Spots 0
Spots 0
Spots 1 I I 2
' Rust 0, zero infection: 1, light infection 1-5 uredinia/m?; 2, heavy infection > 10 uredinia uredinia. a circle of orange-yellow coloured leaf tissue. Leaf surfaces were bubbled and distorted and uredinia were confluent and formed in a ring on both leaf surfaces. Single uredinia formed sub-epidermally and when fully expanded formed large cushions or bubbles on the leaf surface. In vertical section cells underlying the uredinium expanded and divided forming 4-5 layers at the base of the uredinium (Fig. 1).Single uredinia measured 1-2 x 0.5 mm. Uredinia were also formed on stems of young shoots. Uredinia tended to die out by mid summer leaving moribund white cushions on the leaf surface. Surrounding leaf tissue remained healthy and leaves did not fall prematurely. Uredinia of M. coleosporioides, M. epiitea and the Melampsora from 5. viminalis cultivars formed abaxially and were subepidermal (Figs 2-4). Single Uredinia were circular to oblong, and measured 0.125-0.30 mm diam. (Figs 5-7). Urediniospores Shape. Urediniospores of M. epitea, and the Melampsora spp. from 5. viminalis and 5. daphnoidesls. incana x open pollinated cultivars were globose to broadly ellipsoidal. Urediniospores of M. coleosporioides were obovate to pyriform (Figs 8-11). Spore walls of M . epitea, M. coleosporioides and Melampsora
Inoculum ex S. viminalis
Inoculum ex S. incana x daphnoides cv. Tiritea
Spots, necrotic fine spots; odd
1-2 scattered
from S. viminalis were thin (1.4 pm, mean; range 1.0-2.0 pm) whereas walls of Melampsora from 5. daphnoidesls. incana x open pollinated cultivars were thick (2.1 pm mean; range 1.4-3.0 m).Germpores were not evident in urediniospores of the Melampsora from S. viminalis. They were indistinct and partly visible in urediniospores of M. epitea and M . coleosporioides. Germpores were prominent in urediniospores of the Melampsora from 5. daphnoides/S. incana x open pollinated cultivars. Ornamentation. Urediniospores of M. coleosporioides were uniformly echinulate apart from a smooth apical spot. Urediniospores of the other Melampsora rusts were uniformly echinulate (Figs 8-11). Spines of all four Melampsora rusts were finely pointed, with similar profiles. Spines of M . coleosporioides were surrounded by basal annuli (Figs 12-15). Dimensions. The mean length and breadth and wall-thickness dimensions of urediniospores of M. epitea are listed in Table 1, the Melampsora from 5. incana x open pollinated cultivars (Table 2). M . coleosporioides (Table 3); and Melampsora from S. virninalis (Table 4). The coefficients of variation (Table 1-4) were generally less than 5% reflecting the uniform variability of mean length and breadth dimensions between
Studies of Melampsora rusts
Fig. 1. Vertical section through sub-epidermal uredinium of Melampsora ex S. incana on S. incana x daphnoides. Note the 4-5 layers of cells at the base of the uredinium. Also, the expansion and bubbling of the underlying leaf cells. Fig. 2. Vertical section through subepidermal uredinium of M . coleosporioides on S. babylonica. Note the elongate cells bearing the urediniospores and bubbling of the leaf surface. Fig. 3. Vertical section through subepidermal uredinium of Melampsora ex S. viminalis on S. interior. The uredinium is small and the leaf surface is undistorted. Fig. 4. Vertical section through subepidermal uredinium of M . epitea on S. caprea 'PG'. The uredinium is moribund as indicated by the vacuolate cells. hosts within species. The coefficients of variation for wallthickness varied from 5 % for M. 'viminalis' to 17.5 % for M. coleosporioides reflecting the variability of this morphological feature. The range and mean urediniospore length, breadth, wall thickness dimensions and ratios of the four 'species' are presented in Table 6. The mean length and breadth dimensions of the four 'species' were significantly (P < 0.05) different. Only urediniospore walls of the M e b m p sora from S. daphnoidesls. incana x open pollinated cultivars were significantly (P < 0.05) thicker than walls of the other 'species'. The L: B and L : W ratios of M. epifea and Melampsora from S. viminalis were not significantly different (P < 0.05). The L x B and L x B / W dimension of all Melampsora spp. were significantly different (P < 0.05). The mean length and breadth and wall thickness dimensions of the 16 herbarium specimens examined are listed in Table 7. These are further summarised and are presented along with ratios in Table 8. As with field collections, mean length and breadth dimensions of the four Melampsora spp. were significantly different (P < 0.05). The uredinioispore walls of Melampsora from S. daphnoidesls. incana x open pollinated cultivars were significantly thicker (P < 0.05) than other species. The L : B and L : W ratios of M . epitea and the Melampsora from S. viminalis were not significantly different ( P < 0.05). The L x B ratios of the four were significantly different (P < 0.05). The L x B / W ratios of M . epifea and the Melampsora from S. daphnoidesls. incana x open pollinated cultivars were not significantly different (P < 0.05).
Paraphyses. Paraphyses of the four Melampsom rusts were capitate and smooth-walled. Typically paraphyses of the
Melampsora from S. daphnoidesls. incana x open pollinated cultivars were thick-walled (3.0-5.0 pm), those of M . epitea were more variable, some being thin-walled (0.8-1.2 pm), others thick-walled (3.0-5.0 ym) and yet others apically thickened (4.0-5.0 pm). Paraphyses of Melampsom from S. viminalis were mostly thin-walled (1.2 pm and less) with some uniformly thick-walled (2-5-3.0 pm). Paraphyses of M. coleosporioides were uniformly thickened (1.5-2.0 pm), some with apices thickened (up to 5.0 ym). Paraphyses were difficult to measure in view of their variability, particularly the stalks which were easily broken. Accordingly, only the dimensions of the paraphyses head (length, breadth, wall thickness) were measured. These results are summarized in Table 9. Paraphyses heads of all four Melampsom rusts were significantly (P < 0.05) different in size.
Teliospores. Telia were not observed on Salix incana x open pollinated willow cultivars. Telia of the other rusts were single and scattered measuring 0.075-0.125 mm diam. to being confluent in large irreguIarly shaped clumps. Immature telia were amber-brown, turning dark-brown to black on maturity. Telia of M. coleosporioides were amphigenous although principally epiphyllous. They developed subcuticularly, intraepidermally and subepidermally. Telia of M . epifea were mainly hypophyllous and formed intra and subepidermally (Fig. 16).Telia of the Melampsora on S. viminalis cultivars were mainly epiphyllous and formed subepidemally (Fig. 17). Teliospores of M. coleosporioides were significantly (P < 0.05) longer and thinner than those of M. epitea and the Melampsora from S. viminalis, which were not significantly different. Teliospores of M. coleosporioides were rectangular to triangular,
A. G. Spiers and D. H. Hopcroft
1169
Figs 5-7. Fig. 5. Uredinium of Melampsora ex S. viminalis on the adaxial leaf surface of S. inferior. Fig. 6 . Uredinium of M . epifea on abaxial leaf surface of S. caprea 'PG'. Fig. 7. Uredinium of M. cokosporioides on abaxial leaf surface of S. babylonica.
38.6 5 5.1 pm in length (min. 20.0 pm, max. 50.0 pm), by 9.4 f 1.7 pm in breadth (min. 5.0 pm, max. 16.0 pm; Fig. 18). The side walls were 0.8-1.0 pm thick and were slightly thickened (1.5-2.0 pm) apically. Teliospores of M. epifea were also rectangular to triangular 29.8+4.2 pm in length (min. 16.0 pm, max. 40.0 pm) by 11.5 f 1.4 pm in breadth (min. 7.0 pm, max. 16.0 pm). Side walls were uniformly thin (0.5-0.8 pm). Teliospores of the Melampsora from S. viminalis were rectangular to triangular, 31.5 f 5.0 pm in length (min. 20.0 ym, max. 50.0 ym) by 12.4f2.6 pm in breadth (min. 7.0 pm, max. 20.0 pm). Teliospore walls were uniformly thin, measuring 0.5-0.8 pm. Metabasidia of M . coleosporioides were 40-60 pm long and composed of five cells, i.e. a basal cell 20 pm and four sterigmatal bearing cells 8.0-10.0 pm x 5.0-8.0 pm. Sterigmata measured 8.0-12.0-16.0 pm. Mature basidiospores were hyaline, smooth-waIled, apiculate and subgIobose, 6.3 k 0.5 x 5.2k0.5 pm (mean of 50 basidiospores). Metabasidia of M. epifea were 50-60.0 pm being composed of 4 sterigmatal bearing cells, 12.0-16.0 pm x 89-12.0 pm. Sterigmata measured 8.0-16.0-24.0 pm long. Mature basidiospores were hyaline, smooth-walled, apiculate
+
+
and subglobose, 10.5 1.2 x 9.5 1.1 pm (mean of 50 asidiospores). The metabasidium of the Melampsora from S. viminalis was 40.0-50.0 pm with four equally sized, sterigmatal bearing cells, 10.0-13.0 x 7.0-9.0 pm. Sterigmata measure 8.0-12.0 Urn long. Basidiospores were hyaline, smooth-walled, apiculate and subglobose, 9.0 0.6 x 8.7 & 0.9 pm (50 spores). Pycnia and aecia were formed only on Larix decidua plants inoculated with leaves bearing telia of M . epifea. Two pycnia were observed on needles of L. decidua inoculated with leaves bearing telia of M. coleosporioides. However, aecia failed to develop. No pycnia or aecia formed on L. decidua plants inoculated with leaves bearing telia of the Melampsora attacking 5. viminalis.
+
DISCUSSION Melampsora rusts of Salix are notoriously difficult to identify and knowledge of the aecial host is invaluable (Ziller, 1974; Pei ef al., 1993). Two taxonomic systems of willow rust taxonomy are recognized. One system represented by Klebahn (1904) and recognized by Sydow & Sydow (1915) and
Studies of Melampsora rusts
1170
Figs 8-11. Figs 8-10. Globose to broadly ellipsoidal, uniformly echinulate urediniospores of M. epifea (Fig.8), M. ex S. virninalis (Fig. 9 ) and M.ex S. incana x open poll. (Fig.10). Fig. 11. Obovate to pyrifonn, echinulate urediniospores of M. coleosporioides. Note the
partly bald apices on some urediniospores. Gauman (1959) treats rusts which are morphologically similar but with different alternate hosts as separate species. Gaumann (1959) recognized 17 species including a single autoecious species, M . amygdalinae Kleb. pathogenic to Salix friandra and S. pentandra. Within M. larici-epitea six fornzae speciales were recognized according to the Salix host. Pei et al. (1993, 1995) recently followed Gaumann (1959) when describing six Melampsora species from willows in England. An alternative taxonomic system lumped morphologically similar North American and European Melampsora species with different alternate hosts into a collective species, Melampsora epitea (Jorstad, 1940; Wilson & Henderson, 1966). Within M . epifea, two varieties, var. reficulatae (alternate host Saxifraga) and var. epitea were recognized. Within var. epitea were included forms alternating with Saxifraga (Formerly M. arctica); Euonymus (formerly M. euonymi-capraearum);Larix (formerly M . larici-epifea);Orchidaceae (formerly M . repentis); Ribes (formerly M. ribesii-purpureae); Tsuga (M. epifea f. sp. tsugae); Abies (M. abieti-capraearum); Larix (M. bigelowii = M . paradoxa) ( Ziller, 1959, 1974; Arthur, 1962; Wilson & Henderson). Species outside the M . epitea complex include: M . capmearum (alternate host Larix); M . larici-penfandrae (autoecious species); M . salicis-albae and M. allii-fragiles (alternate host Allium) (Wilson & Henderson, 1966).
Parmelee (1989) grouped Arctic willow rusts into M. epitea, hence followed Wilson & Henderson (1966). Ziller (1974) followed the conventional nomenclature of Arthur (1962), stating that critical revision of the group was necessary. Helfer (1992) also urged taxonomic revision of Melampsora on willows. He identified 14 Melampsora species affecting willows in Britain on the basis of spore and sorus characters confirming many species described by Klebahn (1904). Several other Melampsora species have been recorded in Asia including M . coleosporioides (Saccardo, 1905 ; Cummins, 1950; Cummins & Ling, 1950). Walker (1978) and Latch (1980) described M . coleosporioides in Australasia. Criteria used to delimit Melarnpsora species on willows have included the alternate host, apical thickening of aeciospore walls, size, thickening and location of teliospores, morphological features of urediniospores (dimensions, wallthickening, echinulation, germpores) and paraphyses (Arthur, 1962; Wilson & Henderson, 1966; Ziller 1974; Helfer 1992; Pei et. al., 1993, 1995). Morphological criteria used for delimiting Melampsora species on willows need to be identified. More detail is required in descriptions of urediniospores with regard to germpores, wall-thickening and dimensions. Mean length and breadth dimensions (of at least 50 spores) along with standard deviationlstandard error are more use than
A. G. Spiers and D. H. Hopcroft
1171
Figs 12-15. Finely pointed spines on urediniospores of M. epitea (Fig. 1 2 ) ; M. ex S. viminalis (Fig. 13);M. ex S. incana x open poll. (Fig. 14); and M. coleosporioides (Fig. 15). Note the annuli surrounding the spines of M. coleosporioides.
Table 6. Urediniospore dimensions and ratios of Melampsora species from field collections Urediniospore dimensions (pm) Length
M. coleosporioides M. epifea M. ex viminalis M. ex incann x open poll.
Breadth
Wall thickness
Ratios
range
mean
range
mean
range
mean
L:B
L/W
LxB
17.0-260 120-190 13.0-21.0 15.0-22.0
207A1 15.5D 16,OC 17.18
10.&160 10&16.0 11.5-16.0 12.0-19.0
13.7B 13.1D 13.4C 15.6A
1.0-2.0 1-&20 0q2.0 1-4-3.0
1.48 1.4B 1.48 2.1A
1.51A 1.18B 1.198 1.09C
14.80A 11.07B 11.42B 8.14C
283.6A 203.00 214.4C 266.78
Lx B -
W 202-6A 145.0C 153.1B 1270D
Means not followed by the same letter differ significantly (Pi 0.05) according to Duncan's multiple range test.
vague figures usually quoted, i.e. 14-21 x 13-16 ym. The same applies to dimensions of paraphyses, telia and aeciospores. Host range studies are compounded by the difficult taxonomy of Salix species particularly between the Northern and Southern Hemisphere countries. In the present study the influence of the host on urediniospore dimensions was small ( < 4%), and insufficient to obscure rust species differences. There is a need to determine the extent of variability expressed by various morphological features of Melampsora
willow rusts and to decide which features can best be used to differentiate species, that is, the steps 'analysis' and 'synthesis' used by Snyder & Hansen (1954) in their revision of the genus Fusarium.
The present study has shown that morphological features of urediniospores, namely echinulation, length and breadth dimensions, wall thickness, the presence of germpores, and L x B ratios were useful criteria for describing and delimiting species. Urediniospores of M. coleosporioides and the M e l a m -
Studies of Mebmpsora rusts
1172
Table 7. Herbarium collections of willows infected with Mebmpsora spp. examined and dimensions of urediniospores Urediniospore dimensions (pm)
M . c~leos~orioides M . c~leos~orioides M. coleosporioides M . coleosporioides M . ex S.incana x open M . ex S. incana x open M . ex S. incana x open M . ex S. incana x open M . ex S. viminalis M . ex S. viminalis M . ex S. viminalis M . ex S. viminalis M . epiten M. epitea M. epitea M. epitea
poll. poll. poll. poll.
Collection date
Host
Locality
S. babylonica S. babylonica S.babylonica S. babylonica
Whangarei Palmerston North Wanganui Palmerston North Palmerston North Palmerston North Palmerston North Palmerston North Palmerston North Christchurch Palmerston North Palmerston North Palmerston North Palmerston North Turangi Waiuku
N Z 1244 N Z 1009 N Z 1227 NZ 1226 S. interior S. viminalis S.viminalis ' M ' S. viminalis cv. Bowles hybrid S. hippophaefolia S.hippophaefolia 5. cinerea S. cinerea
Length
Breadth
Wall thickness
Means not followed by the same letter differ significantly ( P < 0.05) according to Duncan's multiple range test.
Table 8. Urediniospore dimensions and ratios of Melampsora species from herbarium specimens Urediniospore dimensions (pm)
M . coleo~~orioides M . epitea M. ex S. vimirraiis M. ex S.incana x open poll.
Ratios
Length
Breadth
Wall thickness
21.1A1 15.1D 163C 1758
13.3C 12.9D 14.28 15.9A
1.48 1.48 1.48 2.OA
L:B
L/w
Lx B
LxB W
Means not followed by the same letter differ significantly ( P < 0.05) according to Duncan's multiple range test
Table 9. Urediniospore dimensions and ratios of Melampsora species from herbarium specimens Paraphyses dimensions (pm) Length
M. coleosporioides M . epitea M. ex S. viminalis M . ex S.incana x open poll.
Breadth
Wall thickness
Range
Mean
Range
Mean
Range
Mean
LxB
160-25.0 16.0-34.0 160-300 16.0-35.0
18.1D1 22.6C 23.28 24.3A
12.0-18.0 12.0-24.0 12.0-24.0 12.0-24.0
15.7D 16.6C 18.6A 17.28
08-3.0 08-64 084.4 08-8.0
1.9D 3.08 2.5C 4.8A
284.2D 375.2C 431.5A 41798
Means not followed by the same letter differ significantly (P < 0.05) according to Duncan's multiple range test.
psora from S. daphnoides and S. incana x open pollinated cultivars were morphologically distinct, i.e. the elongate, thinwalled urediniospores of the former compared with the thickwalled, germpore - studded urediniospores of the latter. Urediniospores of Melampsora epitea and the Melarnpsora from S. viminalis were morphologically similar. However with familiarity, the significantly (P < 0.005) smaller, less regular urediniospores of M. epitea, with germpores, were easily distinguished from the regular, germpore free, uniformly thinwalled urediniospores of the Melarnpsora from S. viminalis. Paraphyses were of limited use for species identification. Nevertheless, the small, thin-walled paraphyses of M . coleo-
sporioides were significantly smaller than those of the other Melampsora species. The large paraphyses of the Melampsora attacking S. daphnoides and S. incana x open pollinated cultivars were generally more thick-walled than those of the other Melampsora 'species', although there was much overlap. However, in view of their variability, paraphyses should only be considered as secondary characters for use in species identification. Location of teliospores, both with regard to leaf surface and leaf tissue (subcuticular, intraepidermal, subepidermal) was not a stable morphological feature and therefore not suitable for species identification. Telia of M . epitea mostly formed on
A. G. Spiers and D. H. Hopcroft
Figs 16-18. Fig. 16.Subepidermal, hypophyllous telia of M. epitea. Fig. 17. Subepidermal, amphigenous telia of M ex S. viminniis. Fig. 18. Intraepidermal, epiphyllous telia of M. coleosporioides.
the underside of leaves, whereas telia of the Melampsora attacking S. uiminalis were epiphyllous. Telia of M. coleosporioides were amphigenous. Wilson & Henderson (1966) reported that telia of M. capraearum and M. ribesii-viminalis were epiphyllous, whereas, although telia of M . epifea were mostly hypophyllous, they could also be epiphyllous. Within leaf tissues, telia of M . coleosporioides were sub-cuticular, intraepidermal and subepidermal whereas those of M. epitea and the Melampsora attacking S. viminalis were intraepidermal and subepidermal. No doubt subcuticular telia were also formed. Subcuticular telia were considered to be diagnostic features of M. capraearum and M. ribesii-viminalis by Wilson & Henderson (1966). The thickening (or lack of it) of teliospore walls can be a stable diagnostic feature. In the present study, telia walls were uniformly thin and lacked germination pores. The thickened apical, porate telial wall of M. capraearum was considered to be diagnostic of that species by Wilson & Henderson (1966). Teliospore length and breadth dimensions have not been used to delimit species (Arthur 1962; Wilson & Henderson, 1966), possibly because they are difficult to separate and measure accurately. Teliospores of M. coleosporioides were significantly (P < 0.05) taller and thinner than those of M . epifea and the Melampsora attacking S. uiminalis, which were not significantly different ( P < 0.05).
Morphological features of metabasidia and basidiospores have not been used to characterize Melampsora willow rusts. Ziller (1959) described basidia of M . epifea f. sp. tsugae. In the present study, the metabasidium and basidiospores of M. coleosporioides, M . epitea and the Melampsora attacking S. uiminalis were all significantly different (P > 0.05) in size, particularly the diameter of the metabasidium and the basidiospores, those of M. epifea being almost twice those of M . coleosporioides. Clearly, morphological features and dimensions of metabasidium and basidiospores should be considered, where possible in the description of Melampsora species. Teliospores were not observed on S. daphnoides and S. incana x open pollinated cultivars, yet this rust has overwintered successfully for several years. M . epitea, the Melampsora attacking S. virninalis and M. coleosporioides can also overwinter in the absence of alternate hosts. In the case of the Mehmpsora on S. uiminalis, new uredinia are observed in spring, on the first-formed leaves, I month following budburst. Notwithstanding, infection has been observed to overwinter only on certain cultivars of S. viminalis and never on S. interior. These rusts possibly overwinter as urediniospores or as uredinial mycelium as commonly reported for Melampsora willow rusts (Savile, 1953; Wilson & Henderson, 1966; Ziller, 1974); Parmelee (1989) and Pei ef al., (1995) described a stem-infecting form of M. epitea var. epifea f. sp. hrici-epitea typica attacking S. viminalis which overwintered as urediniospores in buds and stems. M. coleosporioides also overwinters as uredinia on persistent leaves retained on trees. Although telia of this rust germinate profusely in spring they appear to play no role in the life cycle. The alternate host of M. coleosporioides has not been determined (Spiers & Hopcroft, 1988). Melampsora epifea can readily alternate through larch wherever willows and larches are planted together (Spiers & Hopcroft, 1985). In the present study, M. coleosporioides was morphologically distinct from the other Melampsora species and was the only species pathogenic to tree willows. This rust was identified as M . coleosporioides by Walker (1978) and Latch (1980). The rust attacking shrub willows has previously been identified as M. epitea by Dingley (1977). Because the aecial stage of this rust occurs on Larix (Spiers & Hopcroft, 1985), the rust could be classified as M. epitea var. epitea (Wilson & Henderson, 1966), or M. larici-epitea f. sp. fypica (Gaumann, 1959). If the broad species concept of Wilson & Henderson (1966) were followed the Melampsora attacking S. viminalis could also be classified as M. epitea var. epifea (M. epifea sensu J0rstad (1940)). The alternate host of this rust needs to be determined. On the other hand, if Gaumann (1959) was followed the rust would be classified as M. larici-epitea f. sp. fypica. Neither classification is satisfactory since the Melampsora attacking S. viminalis is not wholly morphologically and physiologically similar to M. epitea var. epitea. For example, it has significantly larger (P < 0.05) urediniospores and paraphyses (with thinner walls), epiphyllous telia (cf. hypophyllous) and does not have Larix decidm as an alternate host. Although it shares some Salix hosts with M . epitea var. epifea it tends to be pathogenic to S. viminalis and its hybrids. This rust does not appear to be M. ribesii-viminalis Kleb. because its urediniospores are smaller with thinner walls. Both have thickened paraphyses and
Studies of Melampsora rusts epiphyllous teliospores which are subcuticular in M . ribesiiviminalis (sub-epidermal for New Zealand rust) (Wilson & Henderson, 1966). A race of M . epifea with sub-epidermal teliospores and thickened paraphyses has been recorded on S. virninalis in Europe (Wilson & Henderson, 1966). The Melampsora from S. daphnoides and S. incana x open pollinated cultivars does not fit into the M. epifea complex in view of its thickened urediniospore walls with prominent germspores and large, thick-walled paraphyses. This rust with its large uredinal pustules and distorted leaf surfaces does not fit published descriptions (Jorstad, 1940; Gaumann 1959; Arthur, 1962; Wilson & Henderson, 1966; Ziller, 1974; Parmelee, 1989; Helfer, 1992). In dimensions of urediniospores, wall thickening and germpores it broadly resembles M. capraearum Thiim (Wilson & Henderson, 1966), or M. laricicapraearum Kleb. (Gaumann, 1959). The absence of teliospores hinders confirmation of this. M . larici-epifea f. sp. laricidaphnoides Kleb. has similar urediniospore morphology and willow host range but has telia and alternates via larch (Gaumann, 1959). Clearly, the identity of this rust remains uncertain. It is unusual in that it causes leaf distortion, possibly though the production of growth substances, lacks telia, overwinters in buds and tends to die out by mid summer. How did these Melampsora rusts enter New Zealand? Melarnpsom epifea was first recorded in New Zealand by Dingley (1977) although the rust had been in the country at least 10 years previously (C. W. S. Van Kraayenoord, pers. comm.) Walker (1978) reported that M . epitea had been known in New South Wales and Victoria since 1972. It is unknown whether the rust entered New Zealand from Australia or whether it was introduced independently into both countries. Similarly, it is unknown how the rust attacking S. daphnoides entered New Zealand in 1978. Melampsora coleosporioides entered Australia illegally and was first recorded in April 1978 (Walker, 1978). It crossed the Tasman via wind currents and was recorded in Whangarei, New Zealand in November 1978 (Latch, 1980). The Melampsom attacking S. virninalis appeared briefly on several cultivars in 1981, died out and then reappeared on S. viminalis throughout New Zealand in December 1985. The sudden appearance of this rust throughout New Zealand coincided with that of Marssonina casfagnei which was also recorded on silver poplar throughout New Zealand for the first time in December 1985 (Spiers, 1988). Previously M. casfagnei had entered Australia illegally and was first reported there in February 1984 (J. C. Walker, pers. comm.). The S. viminalis rust is probably also of Australian origin. One possible explanation for the blanket distribution of this Melampsora rust and Marssonina casfagnei throughout New Zealand could be that the fungi were transported via trans-Tasman wind currents which entered New Zealand at the base of and/or top of the country and moved up or down the length of New Zealand depositing conidia and uredinospores. Alternatively several independent trajectories may have been involved. The meteorological aspects of trans-Tasman dispersal have been reviewed by Tomlinson (1973) and Close et al., (1978). This study has shown that the current taxonomy of Melampsora species is inadequate. This problem is overcome at present by lumping difficult to identify species into
1174 complexes like M. larici-epitea (Gaumann, 1957), or M. epifea (Wilson & Henderson, 1966). As suggested by Helfer (1992), a taxonomic revision of Melarnpsora is advocated. We thank Raymond Bennett for the preparation of the electron micrographs.
REFERENCES Ager, A., Ronnberg-Wastljung, A., Thorsen, J. & Siren, G. (1986). Genetic improvement of willows for energy forestry in Sweden. Report No. 43, Swedish University of Agricultural Sciences, Uppsala, Sweden. Arthur, J. C. (1962). hfanual of Rusts rn United States and Cai~ada.Hafner Publ. Co.: New York. Close, R. C., Moar, N. 7.. Tomlinson, A. I. & Lowe, A. D. (1978). Aerial dispersal of biological material from Australia to New Zealand. International Journal of Biometeorology 22. 1-19, Cummins, G. B. (1950). Uredinales of continental China collected by S. Y. Cheo. 1. Mycologia 42. 779-797. Cummins, G. B. & Ling, L. (1950). An index of the plant rusts recorded for continental China and Manchuria. Plant Disease Reporter Supplement 196, 52&556. Dawson, W. M. (1988). Production of biomass from short-rotation coppice willow in Northern Ireland 1974-1987. In Proceedings of the IEA Task I1 Meeting and Workshops on Cell Culture and Coppicing, Oulu, Finland, 24-29 Augwt 1987 (ed. Ari. Ferm). Dingley, J. M. (1977). Additions to the rust fungi of New Zealand. VI. New Zealand Journal of Botany 15, 29-37. Freund, F. J. & Littell, R. C. (1981). SAS for Linear Models. A Guide to tlte Anova and G L M Procedures. SAS Institute Inc.: Cary, North Carolina. Gaumann, E. (1959). Die Rost~ilzeMitteleuropas. Beitrage zur Kryptogamenflora der Schweiz 12. Buchdruckerei Buchler & Co.: Bern. Helfer, S. (1992). The rust diseases of willows in Britain. Proceedings of the Royal Society of Edinburgh 988, 119-134. Jarstad, I. (1940). Uredinales of Northern Norway. Skr$ter Norske Videnskaps Akad. Mat. Nat. KI. 6, 1-45. Klebahn, H. (1904). Die wirtswechseladen Rostpike. Gebriider Borntraeger: Berlin. Latch, B. J. (1980). Weeping willow rust in New Zealand. New ZealandJournal of Agrictcltural Research 23, 535-538. McCracken, A. R. & Dawson, M. (1992). Clonal response in Salix to Melampsora rusts in short rotation coppice plantations. European Journal of Forest Pathology 22, 19-28. Pamelee, J. A. (1989). The rusts (Uredinales) of arctic Canada. Canadian Journal of Botany 67,3315-3365. Pei. M. H., Royle, D. J. & Hunter, T. (1993). Identity and host alternation of some willow rusts (Melampsora spp.) in England. Mycological Research 97, 845-851. Pei, M. H., Royle, D. J. & Hunter, T. (1995). A comparative study of stem- and leaf-infecting forms of Melampsora rust on Salix viminalis in the U.K. Mycological Research 99.357-363. Ronnberg-Wastljung, A. G. & Gunnerbeck, E. (1985). Infection patterns and host differences in Melampsora attacks on Salix ~lantations.Report 38.
Energy Forest project, Department of Ecology and Environmental Research, Swedish University of Agricultural Sciences, Llppsala. Saccardo, P. A. (1905). Sylloge fungorum omnium hucwque cognitorum 17, 265-266. Pavia, Italy. Savile, D. B. 0. (1953). Short season adaptations in the rust fungi. Mycologia 45, 75-87. Schreiner, E. J. (1959). Rating poplars for Melampsora leaf rust infection. Foresty Research Notes, Northeast Experimental Station 90. Snyder, W. C. & Hansen, H. N. (1954). Variation and speciation in the genus Fusarium. Annals of the New York Academy of Sciences 60, 16-23. Spiers, A. G. (1988). Studies of Marssonina castagnei in Australasia. European Journal of Foresf Patkology 18, 65-76.
A. G. Spiers and D. H. Hopcroft Spiers, A. G. & Hathaway, R. L. (1987). Melampsora rusts of Salix in New Zealand. International Energy Agency, Task I1 - Biomass growth and production technology meeting, Oulu, Finland. Uppsala, Sweden. Spiers, A. G. & Hopcroft, D. H. (1985). Ultrastructural studies of the spermat~aland aecial stages of Melampsora larici-populina and Melampsora epitea on Larix decidua. New Zealand Journal of Botany 23, 101-116. Spiers, A. G. & Hopcroft, D. H. (1988). Ultrastructural studies of the telial, basidial, and spermatial stages of the willow rust fungus Melampsora coleosponoides in New Zealand. New ZealandJournal of Botany 26, 423-430. Sydow, P. & Sydow, H. (1915). Monographia Uredinearum 3. Leipzig. Tomlinson, A. I. (1973). Meteorological aspects of trans-Tasman insect dispersal. New Zealand Entomologist 5, 253-268. Van Kraayenoord, C. W. S. (1975). Willows. New Zealand Nature Heritage 7 (98), 2730-2737. Van Kraayenoord, C. W. S., Laundon, G. F. & Spiers, A. G. (1974). Poplar rusts invade New Zealand. Plant Disease Reporter 58, 423-427.
(Accepted 76 February 1996)
1175 Van der Meiden, H. A. (1961). De betekenis van enkek bladzickten van der populier in de populieren-feelt. (The importance of some poplar leaf diseases in poplar culture) Bericht nr. 43. Bosbouwproefstation: Wageningen. Verwijst, T. (1990). Clonal differences in the structure of a mixed stand of Salix viminalis in response to Melampsora and frost. Canadian Journal of Forest Research 20, 602-605. Walker, J. C. (1978). Rust on weeping willow in Australia. Australasian Plant Pathology 7,34. Wilson, M. & Henderson, D. M. (1966). British Rust Fungi. Cambridge University Press: Cambridge U.K. Ziller, W. G. (1959). Studies of Western tree rusts. V. The rusts of Hemlock and Fir caused by Melampsora epitea. Canadian Journal of Botany 37, 109-119. Ziller, W. G. (1974). The tree rusts of Western Canada. Canadian Forestry Service Publication No. 1329.