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Vegetative incompatibility is a fungal allorecognition system characterised by the inability of genetically distinct conspecific fungal strains to form a viable heterokaryon, and is controlled by multiple polymorphic loci termed vic (vegetative incompatibility) or het (heterokaryon incompatibility). We have genetically identified and characterised the first vic locus in the economically important, plant-pathogenic, necrotrophic fungus Botrytis cinerea. A bulked segregant approach coupled with whole genome Illumina sequencing in near-isogenic lines of B. cinerea was used to map a 60-kb genomic region for a vic locus. Within that locus, we identified two adjacent, highly polymorphic open reading frames, Bcvic1 and Bcvic2, which encode predicted proteins that contain domain architectures implicated in vegetative incompatibility in other filamentous fungi. Bcvic1 encodes a predicted protein containing a putative serine esterase domain, a NACHT family of NTPases domain, and several Ankyrin repeats. Bcvic2 encodes a putative syntaxin protein containing a SNARE domain; such proteins typically function in vesicular transport. Deletion of Bcvic1 and Bcvic2 individually had no effect on vegetative incompatibility. However, deletion of the region containing both Bcvic1 and Bcvic2 resulted in mutant lines that were severely restricted in growth and showed loss of vegetative incompatibility. Complementation of these mutants by ectopic expression restored the growth and vegetative incompatibility phenotype, indicating that Bcvic1 and Bcvic2 are controlling vegetative incompatibility at this vic locus.

Sudden decline of the New Zealand cabbage tree (Cordyline australis) results in the rapid death of affected plants within months of first external symptoms becoming apparent. Symptoms, which have been observed in saplings and mature trees, include vascular discoloration and leaf yellowing followed by leaf desiccation and eventual plant collapse. Previous work failed to link the disease with any causal agent. A phytoplasma has now been detected in all symptomatic saplings and some symptomatic trees tested, using one-step and nested polymerase chain reaction (PCR) to amplify portions of the 16S rRNA gene. This phytoplasma was not detected in nonsymptomatic plants. Phytoplasma DNA was found in shoot and rhizome apices, leaves and wood tissue of saplings, and in the rhizome apex and trunk tissues of adult trees. Sequencing of the PCR products from selected samples indicated that the phytoplasma is “Candidatus Phytoplasma australiense.” Phytoplasma cells were detected by transmission electron microscopy in phloem sieve tubes of the rhizomes of affected saplings. One sapling with early symptoms recovered after injection with tetracycline antibiotic, but two saplings with advanced symptoms did not recover. It is concluded that “Candidatus Phytoplasma australiense” is present in symptomatic plants and is the cause of sudden decline.

Historically a single name, Stephanospora flava, was applied to all collections of Stephanospora in Australasia. We used morphological characters with molecular support to differentiate and describe nine novel cryptic species, and refine the circumscription of S. flava. Stephanospora flava is herein restricted to bispored collections from Tasmania, and the quadrisporic Stephanospora tetraspora is raised to species level. Six species (four new) are endemic to Australia, S. flava s.s, S. tetraspora comb. nov., Stephanospora sheoak, Stephanospora cribbae, Stephanospora hystrispora, and Stephanospora occidentiaustralis. Three species Stephanospora poropingao, Stephanospora pounamu, and Stephanospora kanuka are endemic to New Zealand; and one species, Stephanospora aorangi occurs in both Australia and New Zealand. Two other new species, Stephanospora novae-caledoniae and Stephanospora papua, are endemic to New Caledonia or Papua New Guinea, respectively. Analyses of three nuclear gene regions (ITS, ef-1, and LSU) are consistent with current classifications of the family Stephanosporaceae. Athelidium aurantiacum is an outlier, with a strongly supported core of Cristinia (Clade I), Lindtneria (Clade II), Stephanospora, Mayamontana, and Lindtneria trachyspora (Clade III), and a novel lineage of environmental and sporocarp sequences (Clade IV). Taxonomic and nomenclatural issues raised by the presence of both type species of Stephanospora (Stephanospora caroticolor) and Lindtneria (L. trachyspora) in the same clade are discussed.

Species- and population-specific differences in fungicide resistance and aggressiveness within Botrytis makes basic data on genetic diversity important for understanding disease caused by this fungus. Genetic diversity of Botrytis was surveyed between 2008 and 2012 from grapes from five New Zealand wine-growing regions. A total of 1226 isolates were gathered from symptomless flower buds at the start of the growing season and 1331 isolates from diseased fruit at harvest. Two species were found, B. cinerea and B. pseudocinerea. Botrytis pseudocinerea was common in both Auckland vineyards sampled, and infrequent elsewhere. However, even in Auckland, it was rarely isolated from diseased fruit. The presence of the Boty and Flipper transposons was assessed. Isolates with all four transposon states (Boty only, Flipper only, both Boty and Flipper, no transposons) were found for both species. Both vineyards in the Auckland region had high numbers of Flipper-only isolates at flowering; both vineyards from the Waipara region had high numbers of Boty-only isolates at flowering. Most isolates from diseased fruit at harvest contained both transposons. These observations suggest that B. pseudocinerea, and isolates with one or both of the transposons missing, may be less aggressive than B. cinerea, or than isolates with both transposons present. Two clades were resolved within B. pseudocinerea, only one of which has been reported from European vineyards. Phylogenetic diversity within B. cinerea in New Zealand was similar to that known from Europe, including isolates that appear to match Botrytis ‘Group S’. The taxonomic implications of this genetic diversity are discussed.

The genetic variation of 28 populations of Cordyline australis (cabbage tree, tī kōuka) grown from wild-collected seed from across New Zealand was assessed using amplified fragment length polymorphism (AFLP). There was strong geographical structure in the data, with the main differentiation being between plants from the northern North Island and those from the central North Island and further south; a split that was not concurrent with any obvious physical barriers. Further smaller genetic divisions were evident in the data, with eight groups found across the range of C. australis, while genetic variation was found to decrease markedly with latitude. We compare the patterns of genetic variation with earlier studies looking at morphological variation across the range of C. australis, as well as the relative survival of plants from different latitudes in three common garden replicates.

Four species of the sequestrate fungal genus Elaphomyces are reported from New Zealand: Elaphomyces bollardii sp. nov. associated with Leptospermum spp. and Kunzea ericoides, E. luteicrustus sp. nov. associated with Nothofagus menziesii, E. putridus sp. nov. associated with Nothofagus spp., and an unnamed species associated with Nothofagus spp.

Hydrophobins are a remarkable class of small cysteine-rich proteins found exclusively in fungi. They self-assemble to form robust polymeric monolayers that are highly amphipathic and play numerous roles in fungal biology, such as in the formation and dispersal of aerial spores and in pathogenic and mutualistic interactions. The polymeric form can be reversibly disassembled and is able to reverse the wettability of a surface, leading to many proposals for nanotechnological applications over recent years. The surprising properties of hydrophobins and their potential for commercialization have led to substantial efforts to delineate their morphology and molecular structure. In this review, we summarize the progress that has been made using a variety of spectroscopic and microscopic approaches towards understanding the molecular mechanisms underlying hydrophobin structure.

Homologs of the gene encoding the hydrophobin EAS from Neurospora crassa have been identified both in the other conidial species of Neurospora (N. discreta, N. intermedia, N. sitophila, and N. tetrasperma) and selected aconidial species (N. africana, N. dodgei, N. lineolata, N. pannonica, and N. terricola). Southern blot analysis indicated the presence of a single gene in all species examined. EAS-like proteins were purified from the conidial species and each was shown to be the proteolytically processed gene-product of the corresponding eas homolog. While EAS-like proteins were not detected in the aconidial species, putative eas transcripts were detected in some isolates following RT-PCR and the aerial hyphae of these species were hydrophobic. DNA sequences of the coding region of the eas homologs were amplified by PCR and cloned and sequenced from all species except N. pannonica. Phylogenetic analysis of these sequences produced two clusters, the first comprising the conidiating species N. crassa, N. intermedia, N. sitophila, and N. tetrasperma forming a closely related group with N. discreta more distant, and the second comprising the aconidial species N. africana, N. dodgei, N. lineolata forming another closely related group with N. terricola more distant.

Vegetative compatibility has been studied in the kiwifruit (Actinidia spp.) storage rot pathogen Cryptosporiopsis actinidiae by assessing the ability of nitrate non‐utilising mutants generated from the parental strains to complement on medium containing nitrate as sole nitrogen source. A total of 28 strains were allocated to 18 vegetative compatibility groups. One group contained four members, two contained three members, three contained two members, and the remainder contained single members. These numbers are consistent with the independent assortment of five vegetative compatibility genes during sexual recombination. Although a sexual stage has not yet been identified for this fungus, it is concluded that sexual reproduction plays an important role in its biology.

Molecular phylogenetic analyses for the gomphoid-phalloid fungi were conducted based on the five gene dataset with extensive taxon sampling. The monophyly of the gomphoid-phalloid clade was strongly supported, and four well supported major subclades were recognized. Three of the four subclades were represented entirely by gastroid taxa, and only Gomphales contained both gastroid and non-gastroid taxa. While the gastroid morphology is derived from epigeous, nongastroid taxa in Gomphales, the topology of Phallales indicated that truffle-like form is an ancestral morphology of the stinkhorn fruiting bodies. Although basidiospore maturation occurs within the enclosed fruiting bodies of the stinkhorn, the elevation of the mature spore-producing tissue represents an independent origin of the stipe among Basidiomycota. Comparisons are made between previous and new classification schemes, which are based on the results of phylogenetic analyses. Based on the results of these analyses, a new subclass Phallomycetidae, and two new orders, Hysterangiales and Geastrales, are proposed.

Progeny of 28 wild populations of Cordyline australis (cabbage tree, ti kouka) from a 12° latitudinal range in New Zealand grown under uniform garden conditions at Mt Albert, Auckland (36°53'S, 174°43'E), Lincoln, Canterbury (43°38'S, 172°29'E), and Invermay, Otago (45°51'S, 170°23'E), differed in the number of years they took to flower for the first time (years to first flowering, YFF) and in the date their flowers first opened each season (first flowering date, FFD). At Auckland population YFF was unrelated to latitude of origin, but at Lincoln and, especially, Invermay, there was a well‐defined increase in YFF the further north the latitude of origin of populations. FFDs were delayed progressively from Auckland to Invermay and showed a well‐defined pattern of being earlier for southern populations and a weaker trend for earlier flowering with increased altitude of origin. In 2002 and 2003 population FFDs extended over 16–19 weeks at Auckland and narrowed to 2–4 weeks at Invermay. Environmental...

This study reports the molecular characterization of a flexuous rod-shaped mycovirus, Botrytis virus X (BVX), infecting the plant-pathogenic fungus, Botrytis cinerea. BVX contains a ssRNA genome of 6966 nucleotides, and a poly(A) tract at or very near the 3' terminus. Computer analysis of the genomic cDNA sequence of BVX revealed five potential open reading frames (ORFs). ORF1 showed significant amino acid sequence identity to the replicase proteins of plant 'potex-like' viruses, including 73% identity to the RNA-dependent RNA polymerase (RdRp) region of the allexivirus, garlic virus A (GarV-A). The C-terminal region of ORF3 shared amino acid homology with plant 'potex-like' coat proteins. The remaining ORFs did not reveal significant homology with known protein sequences. BVX differs substantially from Botrytis virus F (BVF), another flexuous rod-shaped mycovirus characterized from the same B. Cinerea isolate. It is proposed that the mycovirus BVX belongs to a new, as yet unassigned genus in the plant 'potex-like' virus group, distinct from BVF.

Despite many years of research, fruit loss due to disease is still a major factor in fruit production worldwide. Recent advances in understanding the biology of fruit pathogens, and the complex interactions between host plants and pathogens, offer opportunities for novel ways to better manage these diseases. These opportunities are reviewed with particular reference to the major fruit pathogens (Botrytis, Colletotrichum, Cryptosporiopsis, Monilinia and Venturia) and fruit crops (kiwifruit, apple, grape, avocado and stonefruit) in New Zealand. Approaches based on fungal biology include the possible use of fungal viruses as biocontrol agents, the use of reduced-pathogenicity strains of pathogens as biocontrol agents, and using fungal genomic data and molecular techniques to identify key pathogenicity factors as targets for novel control products. Approaches based on host defence mechanisms include enhancing expression of host defence responses such as antifungal proteins and volatiles through marker-assisted breeding and post-harvest treatments.

The retention of dead leaves covering the trunks of 8‐year‐old trees of Cordyline australis (cabbage tree, ti kouka) was significantly different between 28 wild populations from a 12° latitudinal range in New Zealand when grown under uniform garden conditions at Mt Albert, Auckland (36°53'S, 174°43'E) and Lincoln, Canterbury (43°38'S, 172°29'E). Particularly at Lincoln, trees of populations originating from south of latitude 38°45' mostly had trunks fully covered by dead leaves at the time of observation. Temperatures recorded at Lincoln at heights of 1.40 m and 0.05 m showed that chilling on the trunk surface was reduced by a cover of dead leaves, particularly at 0.05 m during radiation frosts. This effect was separated from the shelter effect of the green leaf tufts of trees and shelter between trees in the experimental plantation. The adaptive function of the retention of dead leaves on the trunk of C. australis in frost‐prone locations in southern New Zealand is considered with reference to o...

Pisolithus is restricted in New Zealand to geothermal areas where it associates with Kunzea ericoides var. microflora (prostrate kanuka) and occasionally Leptospermum scoparium. Here we describe for the first time the ectomycorrhizal morphotypes of three New Zealand Pisolithus species and report the frequency and abundance of these morphotypes against other mycorrhizal fungi associated with these hosts in New Zealand geothermal areas. The three Pisolithus species form typical ectomycorrhizal associations with Kunzea ericoides var. microflora, and one also was observed forming typical ectomycorrhizal associations with Leptospermum scoparium. Although the morphotypes from the three Pisolithus species share many morphological and anatomical characteristics, they vary with regard to the abundance of rhizomorphs. The common occurrence of Pisolithus fruiting bodies at the geothermal sites was matched by frequent and abundant Pisolithus ectomycorrhizas. Pisolithus ectomycorrhizas were frequent (100% of soil cores) and abundant (between 55 and 88% of ectomycorrhizal tips) associates of prostrate kanuka in hot (50 C at 8 cm depth), highly acidic and N depleted soils. The levels of arbuscular mycorrhizal colonization of prostrate kanuka were lower than on K. ericoides and L. scoparium on cooler soils. The stressful conditions where prostrate kanuka dominates probably favor Pisolithus over the mycorrhizal fungi occurring in cooler geothermal areas. Questions about how several genetically similar Pisolithus species co-occur on prostrate kanuka in geothermal areas without mutual competitive exclusion are discussed.

DNA sequence polymorphisms in the putative two-component histidine protein kinase encoded by the Daf1 gene have been identified within a sample of 5 sensitive and 27 dicarboximide-resistant field strains of Botryotinia fuckeliana (anamorph Botrytis cinerea). The gene of 3948 bp is predicted to encode a 1315-amino acid protein comprising an N-terminal region, an amino acid repeat region, which has been hypothesized to be the binding site for dicarboximide fungicide, and a C-terminal region encompassing kinase and response regulator domains. Two amino acid variants were distinguished among the sensitive strains characterized by alanine (group 1), or threonine (group 2), at position 1259 in the C-terminal region. All resistant strains could be classified into either group 1 or group 2 but, in addition, all showed changes in the second amino acid repeat region. On the basis of the differences in this repeat region, four classes of resistant strains were recognized; class 1 characterized by an isoleucine to serine mutation, class 2 by an isoleucine to asparagine mutation, class 3 by an isoleucine to arginine mutation (all at position 365), and class 4 by an isoleucine to serine mutation (position 365) as well as a glutamine to proline mutation (position 369). All classes showed similar low levels of resistance to iprodione and to vinclozolin, except for class 3 and class 4 strains, which show low resistance to iprodione but moderate (class 3) or high (class 4) resistance to vinclozolin. The classes as a group did not differ from sensitive strains in osmotic sensitivity measured as mycelial growth response, but some class 1 strains showed an abnormal morphology on osmotically amended medium. The evolution of the amino acid differences is discussed in relation to field observations. It is proposed that class 1 and class 2 strains arose by single mutations within the sensitive population, whereas classes 3 and 4 arose by single mutations within a resistant population.

A phytoplasma is reported from wild plants of Coprosma robusta, where it is associated with leaf reddening or bronzing and dieback of shoots and branches. It was detected by electron microscopy of phloem tissue, and by one‐step and nested PCR using phytoplasma‐specific primers targeting the 16S rRNA gene. The sequence of the 16S product closely matches that of the Phormium yellow leaf phytoplasma and places the phytoplasma in the putative species "Candidatus Phytoplasma australiense”. It was transmitted by grafting to C. robusta and C. macrocarpa. The symptoms of the disease caused by the phytoplasma (Coprosma lethal decline) in clonally matched graft‐inoculated versus ungrafted cuttings of C. robusta included interveinal chlorosis and abnormal leaf yellowing or reddening, slowing of growth, and shoot dieback. A field survey of C. robusta indicated that shoot dieback and abnormal leaf coloration are common throughout much of New Zealand. "Ca. P. australiense” was detected in some, but not all, sy...

Height growth was measured for 4 years in uniform garden environments at Auckland (36°53'S, 174°43'E), Lincoln, Canterbury (43°38'S, 172°29'E), and Invermay, Otago (45°51'S, 170°23'E) for 28 populations of Cordyline australis from wild sites covering 12° of latitude. Diameters of trunk bases were measured at the end of this period. Populations differed significantly in their final heights and in their growth rates in cool and warm seasons of the year. Generally, northern populations grew best at the Auckland garden site, especially in the warm season. Trunks of southern and higher altitudes were thicker and tree‐height to trunk‐diameter ratios declined the higher the latitude and altitude of population origin. Variation of population trunk diameter was related to phenotypic variation of stem diameter of wild populations. Consideration of the adaptive significance of these results suggests that more rapid and seasonally continuous height growth could confer a competitive advantage in warmer northe...

Summary • Pisolithus is a common ectomycorrhizal (EcM) associate of prostrate kanuka Kunzea ericoides var. microflora (Myrtaceae) in New Zealand geothermal areas. Here, we report the genetic diversity and phylogeny of Pisolithus and interpret the results in relation to the origin of this fungus in New Zealand. • We determined the genetic variation of Pisolithus on the basis of ITS gene sequences and spore morphology. • We identified three Pisolithus species in New Zealand, each matching Australian species associated with eucalypts and acacias. All three species co-occurred locally in thermal areas, with two species sometimes colonizing root tips in the same soil volume, indicating co-occurrence of species on a smaller scale. • We propose that Pisolithus fungi were introduced to New Zealand from Australia by trans-Tasman airflow during recent geological times. The success of this long-distance dispersal of EcM fungi may be related to the capacity of kanuka to act as a ‘nurse plant’ for wind-blown spores.

Nitrate non-utilising (Nit) mutants of six field strains and three single-ascospore strains of Botryotinia fuckeliana were selected by plating mycelial plugs onto a nitrate-containing minimal medium amended with chlorate. Mutants were characterised by growth responses on minimal medium amended with various sole nitrogen sources. For each parental strain two mutants were produced: nit1, defective in nitrate reductase apoenzyme; and NitM, defective in the molybdenum cofactor pathway. Complementation on nitrate minimal medium was observed between nit1 and NitM mutants from the same parent in all instances. However, complementation was not observed between any such combinations when the mutants were derived from different parents. It is concluded that a vegetative incompatibility system operates in B. fuckeliana resulting in multiple vegetative compatibility groups. One of the single-ascospore strains was derived from a cross between two of the field strains, indicating that new compatibility groups can be generated during sexual reproduction. Mycelial interaction zones were formed between all parental strains when they were paired on NaCl-amended medium, indicating congruence between mycelial incompatibility and vegetative incompatibility.

Observations were made on the size, shape, and colour of leaves of 5‐ to 6‐year‐old trees of Cordyline australis (cabbage tree, ti kouka) raised from seed of 28 wild populations from a 12° latitudinal range in New Zealand and grown under uniform garden conditions at Lincoln (43°38'S, 172°43'E). Overall, mean population linear leaf dimensions increased, leaves became broader, and petioles were less defined the further south the population origin. These genotypic patterns were correlated with matching leaf phenotypic characteristics of trees at their source locations. Latitudinally related clines of variation were also shown for leaf colour states and states defining transverse curvature of leaf blades. Blade thickness, purple leaf‐base coloration, and midrib distinc‐tiveness showed regional foci of variation. Character distribution patterns indicate centres of variation in northern North Island and northern South Island. The variations of the leaf character states are discussed in relation to adap...

Winter cold damage in 1996 and 1997 was recorded for 28 populations of Cordyline australis from a wide latitudinal range in New zealand and single populations of C. banskii and C, indivisa grown in field experiments at Lincoln and Invermay New Zealand. Damage to a single plant of C. obtecta grown at Lincoln was also recorded. Cordyline banskii and C. obtecta were damaged the most by freezing. Levels of cold damage to C. australis were closely related to latitude of origin; populations from northern North Island suffered severe damage whereas those from inland southern south Island showed little damage. These relationships were clearest when the populations were exposed to the lowest grass minimum temperature of ‐9.7°C that occurred at Invermay in winter 1996. Retardation of the height growth of the populations most severely damaged in winter 1996 kept them in the frost layer in winter 1997, increasing their exposure to further damage. Variation in cold damage that related to altitude and topograp...

Observations were made on seedlings grown under uniform conditions of 28 wild populations of Cordyline australis (cabbage tree, ti kouka) from a wide latitudinal range in New Zealand and single populations each of C. banksii and C. indivisa. There were highly significant differences in leaf size and shape and in seedling colour between the populations of C. australis. Seedling leaves of C. banksii resembled in shape those of the four C. australis populations north of Auckland and one in north‐west Nelson, suggestive of possible introgression. C. indivisa seedlings differed markedly in leaf size and shape from the other species. The main trend in the genotypic variation of C. australis was clinal along a north‐to‐south latitudinal gradient with seedling leaves becoming longer, narrower, and having a higher frequency of red‐brown coloration the further south the origin of the population. There were populations that deviated significantly from this latitudinal trend, and their locations suggest geog...

To determine whether there are geographical patterns in the phenotypic variation of wild populations of Cordyline australis (ti kouka, cabbage tree), stem size, and leaf size and shape were recorded for populations at 29 locations in New Zealand ranging from latitudes 34°27’ to 46°07'S and altitudes from 1 to 425 m. Correlations between stem and leaf variation and site and stand characteristics were examined. Stem diameter increased with increasing latitude and altitude. Leaf size dimensions and leaf shape ratios that defined differentiation of the petiole, and of leaf blade taper, also showed variation related to latitude. The phenotypic variation was predominantly continuous and followed environmental gradients that could have induced it. There was some local and regional discontinuity of phenotypic variation that was most apparent for stands in central‐eastern North Island.

Selenate-resistant (SelR) mutants of Botrytis cinerea were selected by plating conidia or mycelial plugs onto minimal medium amended with selenate and taurine. Ultraviolet irradiation of conidia to give 5% survival increased mutant yield twenty-fold. Mutants could be divided into three classes based on growth in the presence of selenate or chromate and on response to taurine in minimal media. Nitrate non-utilizing (Nit) mutants, generated as spontaneous sectors on minimal media amended with chlorate, behaved as nit1 mutants in growth tests (i.e. putatively defective in nitrate reductase apoenzyme). When nit1 mutants were paired on medium with nitrate as sole nitrogen source some pairings complemented, behaviour attributed to intragenic complementation. Selected crosses of SelR and nit1 mutants with wild-type strains gave 1[ratio ]1 segregation of both phenotypes and no evidence of linkage to either Mbc1 (benzimidazole resistance) or Daf1 (dicarboximide resistance) markers; loose linkage was confirmed between Mbc1 and Daf1. Strains showing the SelR phenotype may result from mutations in different genes; the genotypic symbol Sel 1 is allocated to one of these mutations. Four-point crosses involving the markers at Sel 1, nit1, Mbc1 and Daf1 generated progeny with all 16 genotypes. Both Sel1R and nit1 mutants were stable following subculture and retained pathogenicity in a bean seedling assay. Complementation was demonstrated between SelR and nit1 mutants selected from the same parent.

A reliable diagnostic method was developed for use in studying the relationship between phormium yellow leaf disease of New Zealand flax (Phormium tenax) and its associated phytoplasma (phormium yellow leaf phytoplasma: PYL). Diagnosis involved a nested PCR (polymerase chain reaction) technique targeting the 16S rRNA gene. DNA was extracted from woody rhizome tissues of NZ flax plants using CTAB and a high salt precipitation step. This method effectively eliminated polysaccharides, gum-like material and other compounds inhibitory to PCRs that occur at high concentrations in diseased NZ flax rhizomes. PCR competence of each DNA preparation from both healthy and yellow leaf diseased plants was assessed using the general prokaryotic 16S rRNA gene primers, Gd1/Berg54. These primers amplified DNA from both diseased and healthy plants. PYL 16S rDNA sequences were not detected consistently following amplification by PCR (35 cycles) using the ‘universal’ phytoplasma-specific primer pairs R16F2/R16R2 or P1/P6. By contrast, PYL was consistently detected in diseased, but not healthy, NZ flax plants, following nested PCR of the products of the above three primer pairs. Nested PCRs involve the primers NGF/NGR, which were designed to hybridize with all phytoplasmas for which published sequences were available. The most sensitive level of detection by nested PCR was achieved using primers R16F2/R16R2, rather than primers P1/P6 or Gd1/Berg54, for the primary amplification step. The consistent association found in this study between yellow leaf disease and PYL further substantiates this phytoplasma as the causal agent. PCR products of the expected size were also amplified by nested PCR using the primers R16F2/R16R2 followed by NGF/NGR from C. roseus tissues infected with five other phytoplasmas representing three distinct phytoplasma groups. Therefore nested PCRs with these pairs of primers should be useful for detecting other phytoplasmas, in particular those occurring at low concentrations or in recalcitrant tissues.

Sequence comparisons and phylogenetic analysis of the 16S rRNA genes and the 16S/23S spacer regions of the phytoplasmas associated with Australian grapevine yellows, papaya dieback and Phormium yellow leaf diseases revealed minimal nucleotide differences between them resulting in the formation of a monophyletic group. Therefore, along with Australian grapevine yellows, the phytoplasmas associated with Phormium yellow leaf and papaya dieback should also be considered as ‘Candidatus Phytoplasma australiense’.

Double-stranded RNA (dsRNA) was demonstrated in mycelial extracts in 143 of 200 isolates of Botrytis cinerea from a range of hosts in New Zealand. The dsRNA profiles differed widely between isolates in the number, size and relative concentration of dsRNA species present. Virus-like particles (VLPs) were observed by electron microscopy in partly purified virus preparations in three of five dsRNA-containing isolates examined. Morphological types present included isometric particles of varying size classes (approx. 30, 35, 40 and 45 nm) and bacilliform particles (approx. 25 × 63 nm). Such particles were not observed in five isolates apparently lacking dsRNA but flexuous rods with a modal length of approx. 720 nm were present in one. Total RNA extraction of partly purified VLP preparations gave similar profiles to those obtained from mycelial dsRNA extractions of the same isolates, suggesting that the VLPs represent encapsidated dsRNA. Comparison of 12 dsRNA-containing and 12 dsRNA-free isolates indicated slight differences between the two groups in radial growth rate, virulence on bean leaves and sclerotial number but the differences were minor and the ranges overlapped.

Three Gallacea species (including G. dingleyae sp. nov., G. eburnea sp. nov.), five Hysterangium species (including H. neotunicatum sp. nov., H. rugisporum sp. nov., H. rupticutis sp. nov., H. youngii sp. nov.), one Phallobata species, and three Protubera species (P. hautuensis sp. nov., P. nothofagi sp. nov., P. parvispora sp. nov.) are recognised in New Zealand. Keys to and descriptions of all taxa are provided.

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38–39 Mb genomes include 11,860–14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to, Author Summary Sclerotinia sclerotiorum and Botrytis cinerea are notorious plant pathogenic fungi with very wide host ranges. They cause vast economic damage during crop cultivation as well as in harvested produce. These fungi are typical examples of necrotrophs: they first kill host plant cells and then colonize the dead tissue. The genome sequences of the two fungi were determined in order to examine commonalities in structure and content and in order to find unique features that may distinguish them from other pathogenic fungi and from saprotrophic fungi. The genomes show high sequence identity and a similar arrangement of genes. S. sclerotiorum and B. cinerea differ in their regulation of sexual reproduction, and the genetic basis and its evolution could be explained from the genome sequence. The genome sequence revealed a striking difference in the number and diversity of secondary metabolism gene clusters, which may be involved in the adaptation to different ecological niches. Altogether, there were no unique features in the genomes of S. sclerotiorum and B. cinerea that could be identified as “silver bullets,” which distinguish these aggressive pathogens from other pathogenic and non-pathogenic fungi. These findings reinforce the quantitative, multigenic nature of necrotrophic pathogenesis.

The mating type of strains of Botrytis cinerea (teleomorph Botryotinia fuckeliana) from a diverse range of hosts and locations throughout New Zealand was determined by crossing with tester strains of known mating type. Most isolates were hetero‐thallic, 24 carrying the MAT 1–1 allele and 17 the MAT1–2 allele, but 9 were homothallic. The mating type alleles were widely distributed with respect to host, district, and fungicide resistance genes. Fungicide resistance phenotypes segregated as distinct characters consistent with the presence of the previously recognised genes Mbcl, conferring resistance to benzimidazole fungicides, and DafI, conferring resistance to dicarboximide fungicides. A new allele is recognised, DafIUR, encoding ultra‐low level dicarboximide resistance not associated with abnormal osmotic sensitivity. Pairing of single ascospore strains on malt extract agar amended with 0.68M NaCl demonstrated a mycelial incompatibility system in this fungus.

Pisolithus is restricted in New Zealand to geothermal areas where it associates with Kunzea ericoides var. microflora (prostrate kanuka) and occasionally Leptospermum scoparium. Here we describe for the first time the ectomycorrhizal morphotypes of three New Zealand Pisolithus species and report the frequency and abundance of these morphotypes against other mycorrhizal fungi associated with these hosts in New Zealand geothermal areas. The three Pisolithus species form typical ectomycorrhizal associations with Kunzea ericoides var. microflora, and one also was observed forming typical ectomycorrhizal associations with Leptospermum scoparium. Although the morphotypes from the three Pisolithus species share many morphological and anatomical characteristics, they vary with regard to the abundance of rhizomorphs. The common occurrence of Pisolithus fruiting bodies at the geothermal sites was matched by frequent and abundant Pisolithus ectomycorrhizas. Pisolithus ectomycorrhizas were frequent (100% of soil cores) and abundant (between 55 and 88% of ectomycorrhizal tips) associates of prostrate kanuka in hot (50 C at 8 cm depth), highly acidic and N depleted soils. The levels of arbuscular mycorrhizal colonization of prostrate kanuka were lower than on K. ericoides and L. scoparium on cooler soils. The stressful conditions where prostrate kanuka dominates probably favor Pisolithus over the mycorrhizal fungi occurring in cooler geothermal areas. Questions about how several genetically similar Pisolithus species co-occur on prostrate kanuka in geothermal areas without mutual competitive exclusion are discussed.

Mycoviruses (fungal viruses) are reviewed with emphasis on plant pathogenic fungi. Based on the presence of virus-like particles and unencapsidated dsRNAs, mycoviruses are common in all major fungal groups. Over 80 mycovirus species have been officially recognized from ten virus families, but a paucity of nucleic acid sequence data makes assignment of many reported mycoviruses difficult. Although most of the particle types recognized to date are isometric, a variety of morphologies have been found and, additionally, many apparently unencapsidated dsRNAs have been reported. Until recently, most characterized mycoviruses have dsRNA genomes, but ssRNA mycoviruses now constitute about one-third of the total. Two hypotheses for the origin of mycoviruses of plant pathogens are discussed: the first that they are of unknown but ancient origin and have coevolved along with their hosts, the second that they have relatively recently moved from a fungal plant host into the fungus. Although mycoviruses are typically readily transmitted through asexual spores, transmission through sexual spores varies with the host fungus. Evidence for natural horizontal transmission has been found. Typically, mycoviruses are apparently symptomless (cryptic) but beneficial effects on the host fungus have been reported. Of more practical interest to plant pathologists are those viruses that confer a hypovirulent phenotype, and the scope for using such viruses as biocontrol agents is reviewed. New tools are being developed based on host genome studies that will help to address the intellectual challenge of understanding the fungal-virus interactions and the practical challenge of manipulating this relationship to develop novel biocontrol agents for important plant pathogens.

The phytoplasma “Candidatus Phytoplasma australiense” has been reported from New Zealand and Australia, where it has been associated with a range of host plants, especially since the 1970s. Partial tuf gene sequences of 36 New Zealand (NZ) isolates from four different host genera revealed nine different variants, which clustered into two distinct groups without any obvious correlation with host or geographic region. Phylogenetic analysis of these sequences, together with those available from Australian isolates, revealed three distinct clades: one found solely in Australia, one found solely in NZ, and a third with representatives from both countries. These divisions are consistent with differences observed in the 16–23S rRNA internal transcribed spacer region; therefore, we conclude that they represent three distinct subgroups: tuf 1, tuf 2, and tuf 3. We estimated a time of divergence for the three clades based on a synonymous substitution rate calculated by comparing the complete tuf gene sequence from the Loofah witches'-broom phytoplasma and “Candidatus Phytoplasma australiense”. Using a calibration date of 110 million years, the estimated time to a common ancestor for all clades (6 to 9 million years ago) suggests divergence during the Miocene, well after the geological separation of NZ and Australia.

A novel glycoside, (E)-4-hydroxy-2-(hydroxymethyl)but-2-ennyl β- d -allopyranoside (1, cardiomanol) has been identified as the major soluble carbohydrate in the leaves of Cardiomanes reniforme. The structure of 1 was established by chemical and spectroscopic methods.

Phytophthora Clade 5 is a very poorly studied group of species of oomycete chromists, consisting of only two known species P. castaneae (≡ P. katsurae, nom. illegit .) and P. heveae with most isolates from East Asia and the Pacific Islands. However, isolates of two important disease-causing chromists in Clade 5, one of kauri ( Agathis australis ) in New Zealand, the other of coconut ( Cocos nucifera ) in Hawaii, poorly match the current species descriptions. To verify whether these isolates belong to separate species a detailed morphological study and phylogenetic analysis consisting of eight genetic loci was conducted. On the basis of genetic and morphological differences and host specificity, we present the formal description of two new species in Clade 5, Phytophthora agathidicida sp. nov . and Phytophthora cocois sp. nov . To clarify the typification of the other Clade 5 species, an authentic ex-holotype culture of Phytophthora castaneae is designated and P. heveae is lectotypified and epitypified.

Two plasmids from the plant-pathogenic mollicute “Candidatus Phytoplasma australiense” were completely sequenced from two isolates derived from different plant hosts. Plasmid pPAPh2 (3607 bp) was obtained from Phormium showing Phormium yellow leaf symptoms and pPASb11 (3635 bp) from strawberry showing strawberry lethal yellows symptoms. The plasmids varied in their copy number and nucleotide sequence yet contained the same four open reading frames (ORFs). The deduced amino acid sequence derived from ORF1 shares similarity with hypothetical proteins encoded on the plasmids from onion yellows and beet leafhopper-transmitted virescence agent phytoplasmas. The deduced amino acid sequences of both ORF2 and ORF3 share similarity with functionally unknown proteins on the chromosome of onion yellows phytoplasma. An ORF with a similar sequence to ORF2 is also present on the chromosome of “Ca. P. australiense.” The deduced amino acid sequence derived from ORF4 is most similar to replication proteins encoded by other phytoplasma plasmids and by geminiviruses, the only protein on the plasmids for which a putative function can be assigned. The identities of the deduced amino acid sequences of ORF1, ORF2, ORF3, and ORF4 between pPAPh2 and pPASb11 were 89, 68, 91, and 68%, respectively; the differences being consistent with the subgroup status of the parental phytoplasmas.

Controlled pollination among four individuals of Cordyline australis (cabbage tree, ti kouka) demonstrate that this species is self-incompatible, setting little or no seed when self-pollinated. Seed production per fruit in wild trees was less than half that observed following artificial pollination. Parthenocarpic fruit production is recorded in some wild populations.

A two-component histidine protein kinase gene, homologous to os-1 from Neurospora crassa, was cloned and sequenced from a single ascospore isolate of Botryotinia fuckeliana. A series of nine spontaneous mutants resistant to dicarboximide fungicides was selected from this strain and characterized with respect to fungicide resistance and osmotic sensitivity. Genetic crosses of the mutants with an authentic Daf1 strain showed that the phenotypes mapped to this locus. Single point mutations (seven transitions, one transversion, and one short deletion) were detected in the alleles of the nine mutants sequenced. The mutational changes were shown to cosegregate with the dicarboximide resistance and osmotic sensitivity phenotypes in progeny obtained from crossing selected resistant strains with a sensitive strain. All mutations detected are predicted to result in amino acid changes in the coiled-coil region of the putative Daf1 histidine kinase, and it is proposed that dicarboximide fungicides target this domain.

This study reports the first sequence of a flexuous rod-shaped mycovirus and also the first molecular characterization of a virus that infects the plant-pathogenic fungus Botrytis cinerea. The mycovirus Botrytis virus F (BVF) contains an ssRNA genome of 6827 nucleotides and a poly(A) tract at or very near the 3′ terminus. Computer analysis of the genomic cDNA sequence of BVF revealed two potential open reading frames (ORFs) encoding proteins of 212 kDa (ORF1) and 32 kDa (ORF2). ORF1 showed significant sequence identity to the RNA-dependent RNA polymerase (RdRp)-containing proteins of plant ‘tymo-’ and ‘potex-like’ viruses. However, the ORF1 protein contained an opal putative readthrough codon between the helicase and RdRp regions, a feature not seen in this position in ‘tymo-’ and ‘potex-like’ replicases sequenced to date. ORF2 shared amino acid similarity with coat proteins of plant ‘potex-like’ viruses. Three untranslated regions were present in the genome, comprising a region of 63 nucleotides preceding the initiation codon of ORF1, a 93 nucleotide stretch between ORFs 1 and 2 and a 3′-terminal region of 70 nucleotides preceding the poly(A) tract. The nucleotide sequence of a putative defective RNA (D-RNA) of 829 nucleotides was also determined. The D-RNA contained one potential ORF comprising the N-terminal region of the replicase fused in-frame to the C-terminal region of the coat protein. It is proposed that the mycovirus BVF belongs to a new, as yet unassigned genus in the plant ‘potex-like’ virus group.

Background ‘Candidatus Phytoplasma australiense’ is associated with at least nine diseases in Australia and New Zealand. The impact of this phytoplasma is considerable, both economically and environmentally. The genome of a NZ isolate was sequenced in an effort to understand its pathogenicity and ecology. Comparison with a closely related Australian isolate enabled us to examine mechanisms of genomic rearrangement. Results The complete genome sequence of a strawberry lethal yellows (SLY) isolate of ‘Candidatus Phytoplasma australiense’ was determined. It is a circular genome of 959,779 base pairs with 1126 predicted open reading frames. Despite being 80 kbp larger than another ‘Ca. Phytoplasma australiense’ isolate PAa, the variation between housekeeping genes was generally less than 1% at a nucleotide level. The difference in size between the two isolates was largely due to the number and size of potential mobile units (PMUs), which contributed to some changes in gene order. Comparison of the genomes of the two isolates revealed that the highly conserved 5′ UTR of a putative DNA-directed RNA polymerase seems to be associated with insertion and rearrangement events. Two types of PMUs have been identified on the basis of the order of three to four conserved genes, with both PMUs appearing to have been present in the last common ancestor of ‘Ca. Phytoplasma asteris’ and ‘Ca. Phytoplasma australiense’. Comparison with other phytoplasma genomes showed that modification methylases were, in general, species-specific. A putative methylase (xorIIM) found in ‘Ca. Phytoplasma australiense’ appeared to have no analogue in any other firmicute, and we believe has been introduced by way of lateral gene transfer. A putative retrostransposon (ltrA) analogous to that found in OY-M was present in both isolates, although all examples in PAa appear to be fragments. Comparative analysis identified highly conserved 5′ and 3′ UTR regions of ltrA, which may indicate how the gene is excised and inserted. Conclusions Comparison of two assembled ‘Ca. Phytoplasma australiense’ genomes has shown they possess a high level of plasticity. This comparative analysis has yielded clues as to how rearrangements occur, and the identification of sets of genes that appear to be associated with these events.

Phormium yellow leaf (PYL) phytoplasma causes a lethal disease of the monocotyledon, New Zealand flax (Phormium tenax). The 16S rRNA genes of PYL phytoplasma were amplified from infected flax by PCR and cloned, and the nucleotide sequences were determined. DNA sequencing and Southern hybridization analysis of genomic DNA indicated the presence of two copies of the 16S rRNA gene. The two 16S rRNA genes exhibited sequence heterogeneity in 4 nucleotide positions and could be distinguished by the restriction enzymes BpmI and BsrI. This is the first record in which sequence heterogeneity in the 16S rRNA genes of a phytoplasma has been determined by sequence analysis. A phylogenetic tree based on 16S rRNA gene sequences showed that PYL phytoplasma is most closely related to the stolbur and German grapevine yellows phytoplasmas, which form the stolbur subgroup of the aster yellows group. This phylogenetic position of PYL phytoplasma was supported by 16S/23S spacer region sequence data.

Templeton, M. D., Greenwood, D. R., and Beever, R. E. 1995. Solubilization of Neurospora crassa rodlet proteins and identification of the predominant protein as the proteolytically processed eas (ccg-2) gene product. Experimental Mycology 19, 166-169. Proteins from conidial rodlet preparations of Neurospora crassa were solubilized in trifluoroacetic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized rodlets revealed a predominant protein of approximately 7 kDa. This protein was absent from preparations of N. crassa cultures carrying the eas mutation. The protein was purified by reverse-phase high-performance liquid chromatography and the N-terminal amino acid sequence of the purified protein was found to be identical to an internal portion of the deduced amino acid sequence of eas. Comparison of the sequences indicates a 29-amino-acid leader which is cleaved to generate the mature protein.

Kauri illthrift commonly known as Kauri dieback has been identified as an increasing problem affecting kauri (Agathis australis) across the Auckland region A water and soilborne pathogen Phytophthora taxon Agathis (PTA) has been identified as a causal agent of Kauri dieback at some locations particularly within the Waitakeres Ranges Regional Park and Great Barrier Island PTA is associated with a collar rot causing large bleeding basal lesions yellowing foliage and tree death A range of other causal agents including Phytophthora cinnamomi and environmental stress factors were also associated with symptoms at many sites In 2008 Auckland Regional Council implemented a range of standard operational procedures to manage the disease across the region Surveillance surveys are underway to assess overall tree health as well as the potential distribution of PTA in Aucklands kauri forests Survey sites were prioritised in areas with high conservation value iconic trees or high levels of soil disturbance such as tracks intersecting kauri root zones Risk management of the suspected primary vectors of the PTA including people and feral pigs is underway

Considering New Zealand’s small size and remoteness, a remarkable number of plant pathogens have been recorded. A complete review of the records of plant viruses, viroids and mollicutes in New Zealand found good documentary evidence for 186 viral diseases, 5 viroids and 6 mollicutes, of which 59 viruses, 3 viroids, and 4 mollicutes have been recorded since publication of the previous complete review in 1989. Based on a lack of sufficient documented evidence, a further 27 viruses, 5 viroid and 3 mollicute records were considered unconfirmed. The confirmed pathogens not only infect agricultural and horticultural crops but some also affect the native flora.

Elsinoe dracophylli sp. nov. is described and illustrated from Dracophyllum pyrimidale and D. traversii. It is the first Elsinoe species to be described from a member of the Epacridaceae, and is distinguished from the Ericaceae-inhabiting species by ascospore size.

Competition was assessed between aggressive and non-aggressive strains of Botrytis cinerea on French bean (Phaseolus vulgaris) leaves by inoculating strains simultaneously and sequentially (6, 12 or 24 h later). The lesions produced when an aggressive strain (forming large lesions) and a non-aggressive strain (forming small, restricted lesions) were inoculated simultaneously were intermediate in size between those produced by the strains when inoculated individually. suggesting that the two strains were co-existing in the lesion. However, in sequential inoculation when the second inoculation was delayed 6 h or more, lesion sizes typical of the first inoculated strain were found indicating that the first strain to be inoculated dominated the infection site. Competition between two aggressive strains, individually identifiable by genetic markers, was found to follow a similar pattern, suggesting that the dominating effect of the first strain is a general phenomenon. Domination of the lesion site by the nonaggressive strain prevented an aggressive strain from forming spreading lesions suggesting a possible use for these strains as a biocontrol agent in some disease situations.

Phonnium yellow leaf (PYL) phytoplasma was transmitted from diseased to healthy New Zealand flax (Phormium tenax) by the native planthopper, Oliarus atkinsoni (Homoptera: Cixiidae). By contrast, transmission was not effected by the introduced passionvine hopper, Scolypopa australis (Homoptera: Ricaniidae). Successful transmission of PYL phytoplasma from New Zealand flax to New Zealand flax by O. atkinsoni was demonstrated by symptomatology and by polymerase chain reaction (PCR) of the test plants using phytoplasma-specific primers to the 16s rRNA genes. When the salivary glands and the remaining body of the planthoppers used in the transmission studies were tested separately by PCR for the presence of phytoplasma, PYL phytoplasma was detected in 100% of both the salivary glands and the bodies of pre-transmission O. atkinsoni, and in 44% and 67% of the salivary glands and the bodies of post-transmission planthoppers, respectively. The phytoplasma was not detected by PCR in the whole bodies of hoppers of S. australis.

Nitrogen non-utilising mutants were recovered from 32 strains of Colletotrichum gloeosporioides (Glomerella cingulata) isolated from h its of apple and other species. Mutants putatively defective in the nitrate reductase structural gene were recovered from all strains, and mutants putatively defective in molybdenum-containing cofactor synthesis from 17 strains. Complementation tests between these two classes of mutants on a medium containing nitrate as sole nitrogen source indicated the presence of at least 11 distinct vegetative compatibility groups, including at least six from apple.