Passalora perplexa, an important pleoanamorphic leaf blight ... - CBS

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BEILHARZ ET AL.unable to find any combination of synanamorphs thatwould equate with the cercosporoid fungus infectingphyllodes of A. crassicarpa and described here. Thecorrect or most logical means of treating the nomenclatureof pleoanamorphic fungi remains somewhatsubjective, and has not been defined in the Code ofBotanical Nomenclature. The well-known genericname Passalora Fr. sensu Crous & Braun (2003) iscorrect for the hyphomycetous synanamorph, and it isparticularly useful in this case because most coloniesof the fungus show liberal and conspicuous sporulationof this morph, whereas the cryptic Type 2 synanamorphis less likely to be observed, and isunlikely to be found in isolation. Gams (1982) suggestedthat the anamorph form with the greatest differentiationshould have priority (unless it is rare), aview which further supports the application of thename Passalora to the fungus on A. crassicarpa.The conidia of the Type 1 and Type 2 synanamorphsof the cercosporoid fungus from A. crassicarpa,although easily distinguished, show somesimilarity in morphology. The Type 2 conidia aresomewhat cercosporoid in type and reminiscent ofsome species of Colletogloeum Petr., but the conidiomatado not fit with the acervular conidiomata of thatgenus. Critical differences between the two types ofconidia, including pigmentation, can be linked to theirrelative positions in relation to the host tissue. Forexample, the thickened hila of Type 1 conidia areprobably associated with their readiness to secede(Beilharz 1994). In contrast, the broader, unthickenedhila of Type 2 conidia are more appropriate to passiverelease following breakdown of overlying fungal andhost tissues.Acacia crassicarpa is a species that has becomeincreasingly important in plantations in various partsof South-East Asia, where it is grown specifically forthe production of pulp. The relatively recent outbreakof a serious leaf blight disease caused by a cercosporoidfungus has demanded an appropriate taxonomictreatment of this organism. This study represents acollaborative effort by a number of research groupswho have an interest in this fungus and the diseasewith which it is associated. Here, we describe a potentiallydevastating, newly recognised disease of A.crassicarpa, and describe the causal organism, a novelpleoanamorphic species of Passalora.MATERIALS AND METHODSIsolatesAt VPRI, cultures were derived from the Australianspecimen VPRI 21125 by the following means. Naturally-producedType 1 conidia were lifted from lesionsen masse with a fine needle and jab-inoculated on to 2% potato-dextrose agar (PDA; Difco) plates emendedwith achromycin (0.05 mg/mL) (PDA+A). Similarly472harvested Type 1 conidia were also suspended in adrop of water containing a trace of Tween 80, streakedout on to 2 % tap water agar and transferred individuallyto PDA+A the following day, after germinating.Type 2 conidia formed in vitro in PDA cultures derivedfrom individual Type 1 conidia were used toprovide single-conidial isolates as described above. Inaddition, whole, pale, smooth protuberant stromatalacking external conidiophores and putatively containingType 2 conidia, were lifted from the phyllodesurface with a fine, sterile needle and placed directlyonto PDA+A. All PDA+A cultures were transferred toPDA after 3–7 d and grown on for up to 2 mo in thedark at 22 °C. The choice of the various forms ofinoculum was determined by the ease with which theycould be harvested from infected phyllodes or cultures.For example, Type 1 conidia were abundant onthe natural subtrate, but occurred in comparativelysmall numbers in culture, where they were liable to becontaminated with Type 2 conidia. Type 2 conidiawere abundant in wet masses in culture; in nature,however, they tended to remain aggregated and oftencould not be completely freed from excised conidiomata,despite the application of pressure on coverslipsor attempts to tease the elements apart in a drop ofwater on a microscope slide. Type 3 conidia were notseen in 2-mo-old cultures on PDA, and Type 2 conidiafrom these cultures germinated normally on fresh agarplates. The Australian specimens and a dried cultureof VPRI 21125 have been deposited in herb. VPRI,Knoxfield, Victoria, Australia.At CBS, single Type 1 conidial isolates werederived from Indonesian specimens and cultivated on2 % malt extract agar (MEA; Difco) as described byCrous (1998). Colonies sporulated on MEA after 1–2mo incubation on the laboratory bench in daylight atroom temperature, forming conidiomata containingType 1 and Type 2 conidia. After 3 mo incubation onMEA, conidiomata with Type 2 conidia were observedto also give rise to Type 3 conidia, a formobserved only in culture. Specimens and cultures havebeen deposited in the herbarium and culture collectionof CBS in Utrecht, the Netherlands.A phylogeny of the cercosporoid fungi occurringon Acacia, including Passalora perplexa, is presentedelsewhere in this volume (Crous et al. 2004).MorphologySlide preparations were made in lactic acid and 50examples of each structure were measured under a×100 oil immersion lens using Olympus BH–2 (VPRI)or Zeiss Axioskop (CBS) light microscopes. The 95 %confidence intervals were also determined for conidialdimensions, with the extremes in conidium length andwidth given in parentheses. Colony colour was determinedon 2 % MEA after 3 mo at 25 °C in the darkusing the colour designations of Rayner (1970).
PASSALORA PERPLEXA AND ITS SYNANAMORPHSFigs 1–6. Passalora perplexa. 1. Trees showing defoliation. 2–6. Symptoms associated with Crassicarpa leaf blight.RESULTSDisease symptomsLesions occur primarily on the phyllodes of A. crassicarpabut they can also form on the petioles andyoung shoots. Phyllode lesions are initially small andtypically elliptical, and are surrounded by a distinctchlorotic halo (Figs 1–6). On freshly formed lesions,fascicles of grey-brown conidiophores and denseolivaceous spore masses can easily be seen. Lesionsformed at the edges of phyllodes or abutting primaryveins can cause severe malformation and curling ofthe phyllodes (Figs 2–6). Infections are often severe,causing the dramatic malformation of the apicalportions of young (1–2-yr-old) trees (Fig. 1).TaxonomySequences obtained for the ITS region in the laboratoriesof both VPRI and CBS, confirmed that the Australianand Indonesian specimens represented the sametaxon. The DNA sequence analyses also showed thatthe fungus is an anamorph of Mycosphaerella, clusteringwith Cercospora loranthi McAlpine (Crous et al.2004, fig. 1), which is a species of Passalora. Theserelationships have been discussed elsewhere (V.C.Beilharz, in press). Sequences of P. perplexa havebeen deposited in GenBank, and the alignment ofsequence data in TreeBASE (Crous et al. 2004).The habit, morphology, pigmentation and scarcharacteristics of Type 1 conidiophores and conidiaare characteristic of the genus Passalora. This observationis consistent with the results of the DNA-basedcomparisons. Currently there are no species of Passaloraknown from Acacia (Crous & Braun 2003), andhence this species with its pigmented Type 1 conidiaand thickened, darkened, refractive conidial hila canbe described as new. Prior to the discovery of additionalcoelomycetes resembling the Type 2 synanamorphand their affiliations being established, it wouldbe inappropriate to provide a separate generic namefor the Type 2 synanamorph or to name this synanamorph.Passalora perplexa Beilharz, Pascoe, M.J. Wingf.& Crous, sp. nov. MycoBank MB500123. Figs7–27.Etymology: Named because of the unusual combinationof conidial synanamorphs.Fungus pleoanamorphicus conidia generis Passalorae etcoelomycitica formans. Conidiophora solitaria vel laxeaggregata, pallide vel medio-brunnea, levia vel verruculosa,subcylindrica, ramosa vel simplicia, pluriseptata, sympodialiterproliferentia, 15–80(–116) µm longa, 3–5 µm lata.Cellulae conidiogenae terminales, verruculosae vel rugosae,simplices, subcylindricae, apicem rotundatum versusangustatae, 15–20 × 3–4 µm; cicatrices modice inspissataeet fuscatae, refringentes, 1–2 µm diam. Conidia solitaria,pallide olivacea vel medio-brunnea, levia vel eximie verruculosa,recta vel curvata, anguste obclavata vel subcylindri-473
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Passalora perplexa, an important pleoanamorphic leaf blight ... - CBS

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