Pisolithus is an important ectomycorrhizal genus world-wide, however to date we remain largely ignorant of the genetic and functional variation that exists within isolates of this genus. Fifty-three isolates of Pisolithus were obtained from various locations in central and eastern Australia and genetic variation within the isolates was assessed using ITS-RFLP and ITS sequencing analyses. RFLP analysis initially grouped the isolates into eight RFLP types. Neighbour-joining analysis of ITS sequences with Pisolithus ITS sequences available in databases clustered the majority of isolates into four groups within two major clades, each comprising isolates of similar basidiospre characteristics. Most Australian isolates correspond with recent provisional descriptions of P. albus or P. marmoratus. One isolate (LJ30) had low sequence identity (61.6-78.0%) to the other isolates and probably represents a separate undescribed Australian species. Significant intraspecific variation was observed in ITS-RFLP profiles for the putative P. albus isolates, suggesting that the sole use of RFLP analysis in diversity assessment may over-estimate Pisolithus species richness. Investigations were also initiated to identify if a relationship exists between genetic and physiological diversity in Australian Pisolithus. It is, however, clear that extensive physiological variation exists in Australian Pisolithus isolates. The size and distribution of genets of Australian Pisolithus species I and II ( putative P. albus and P. marmoratus) was also assessed using microsatellite-primed PCR to gain a better understanding of the likely distribution of underground mycelial networks and possible reproduction strategies in native soils. The data demonstrate that both species have the ability to be long-lived and extend for significant distances in native soils in undisturbed conditions. The field site for Pisolithus species I, however, also contained of a large number of small individuals suggesting that this species may employ a life-history strategy combining r-, C and S characteristics depending on local soil conditions / Doctor of Philosophy (PhD)

Pisolithus is an important ectomycorrhizal genus world-wide, however to date we remain largely ignorant of the genetic and functional variation that exists within isolates of this genus. Fifty-three isolates of Pisolithus were obtained from various locations in central and eastern Australia and genetic variation within the isolates was assessed using ITS-RFLP and ITS sequencing analyses. RFLP analysis initially grouped the isolates into eight RFLP types. Neighbour-joining analysis of ITS sequences with Pisolithus ITS sequences available in databases clustered the majority of isolates into four groups within two major clades, each comprising isolates of similar basidiospre characteristics. Most Australian isolates correspond with recent provisional descriptions of P. albus or P. marmoratus. One isolate (LJ30) had low sequence identity (61.6-78.0%) to the other isolates and probably represents a separate undescribed Australian species. Significant intraspecific variation was observed in ITS-RFLP profiles for the putative P. albus isolates, suggesting that the sole use of RFLP analysis in diversity assessment may over-estimate Pisolithus species richness. Investigations were also initiated to identify if a relationship exists between genetic and physiological diversity in Australian Pisolithus. It is, however, clear that extensive physiological variation exists in Australian Pisolithus isolates. The size and distribution of genets of Australian Pisolithus species I and II ( putative P. albus and P. marmoratus) was also assessed using microsatellite-primed PCR to gain a better understanding of the likely distribution of underground mycelial networks and possible reproduction strategies in native soils. The data demonstrate that both species have the ability to be long-lived and extend for significant distances in native soils in undisturbed conditions. The field site for Pisolithus species I, however, also contained of a large number of small individuals suggesting that this species may employ a life-history strategy combining r-, C and S characteristics depending on local soil conditions / Doctor of Philosophy (PhD)

La microflore des raisins est importante d'un point de vue technologique car elle conditionne en partie la qualité du vin. Or, la diversité des flores fongiques présentes sur baies de raisin ainsi que leur potentiel de contamination du produit final ne sont pas encore pleinement connus. Dans ce cadre, la caractérisation des flores fongiques cultivables présentes sur baies de raisin a été réalisée par PCR ITS-RFLP. 41 espèces de moisissures différentes sur les 43 étudiées appartenant à 11 genres différents ont été caractérisées de façon fiable. Seules les espèces Penicillium thomii et Penicillium glabrum ont présenté le même profil. Ainsi 96.3% des souches étudiées ont été caractérisées avec au maximum 4 enzymes de restriction et 41.5% des souches ont pu l'être avec seulement 2 enzymes de restriction. Ces résultats ont permis d'enrichir les bases de données, moyennement pourvues en séquences ITS caractéristiques de genres ou d'espèces de moisissures présentes sur baies de raisin. De plus, une étude exhaustive des moisissures présentes sur baies de raisin en Bourgogne a permis, par PCR ITS-RFLP, d'identifier 199 souches au niveau de l'espèce et ce quelque soit le genre. Penicillium spinulosum est l'espèce majoritaire isolée pour le millésime 2008 en Bourgogne. Parallèlement, la quantification de Botrytis cinerea, choisi comme micro-organisme modèle, a été réalisée par qPCR. La technique qPCR décrite dans ce travail présente (i) une bonne sensibilté avec une limite de détection de 6.4 pg d'ADN correspondant à 540 spores, (ii) l'originalité de travailler en échantillons naturellement contaminés et la fiabilité d'utiliser un standard interne. L'évaluation de l'efficacité de différentes stratégies de traitements anti-Botrytis a confirmé l'importance de la prophylaxie (effeuillage) dans la lutte contre Botrytis cinerea.

La microflore des raisins est importante d’un point de vue technologique car elle conditionne en partie la qualité du vin. Or, la diversité des flores fongiques présentes sur baies de raisin ainsi que leur potentiel de contamination du produit final ne sont pas encore pleinement connus. Dans ce cadre, la caractérisation des flores fongiques cultivables présentes sur baies de raisin a été réalisée par PCR ITS-RFLP. 41 espèces de moisissures différentes sur les 43 étudiées appartenant à 11 genres différents ont été caractérisées de façon fiable. Seules les espèces Penicillium thomii et Penicillium glabrum ont présenté le même profil. Ainsi 96.3% des souches étudiées ont été caractérisées avec au maximum 4 enzymes de restriction et 41.5% des souches ont pu l’être avec seulement 2 enzymes de restriction. Ces résultats ont permis d’enrichir les bases de données, moyennement pourvues en séquences ITS caractéristiques de genres ou d’espèces de moisissures présentes sur baies de raisin. De plus, une étude exhaustive des moisissures présentes sur baies de raisin en Bourgogne a permis, par PCR ITS-RFLP, d’identifier 199 souches au niveau de l’espèce et ce quelque soit le genre. Penicillium spinulosum est l’espèce majoritaire isolée pour le millésime 2008 en Bourgogne. Parallèlement, la quantification de Botrytis cinerea, choisi comme micro-organisme modèle, a été réalisée par qPCR. La technique qPCR décrite dans ce travail présente (i) une bonne sensibilté avec une limite de détection de 6.4 pg d’ADN correspondant à 540 spores, (ii) l’originalité de travailler en échantillons naturellement contaminés et la fiabilité d’utiliser un standard interne. L’évaluation de l’efficacité de différentes stratégies de traitements anti-Botrytis a confirmé l’importance de la prophylaxie (effeuillage) dans la lutte contre Botrytis cinerea. / Microbial population of grapes is important from a technological point of view because it determines the quality of wine. But few studies have focused on fungal populations of grapes. A better knowledge of the fungal diversity on grapes, particularly as concerns species responsible for wine defects, may help efforts to control their development.We report the development of a PCR ITS-RFLP method as a fast and easy technique for identifying species of fungal genera present on grapes. By this methode, 41 different fungal species among 43 studied species belonging to 11 genera were characterized at the species level. Only P. thomii remained indistinguishable from P. glabrum. Using this PCR-ITS-RFLP, 96.3% strains tested could be differentiated to the species level with only four enzymes and 41.5% only with two enzymes. Moreover this work has contributed to the enriching of the database of fungal ITS sequences.Thus 199 isolated strain were on grapes in Burgundy vineyard were chacacterized at species level indepdantly of the genus by this method. P. spinolusum was the most frequently isolated species of Penicillium in Burgundy for 2008 vintage. Paralelly, the quantification of Botrytis cinerea, used as model, was developped by qPCR. The assay contained an internal amplification control to compensate for variations in DNA extraction and the various compounds from grapes, had high efficiency and the limit of detection was estimated to be 6.3 pg DNA (corresponding to 540 spores). This method was applied to assess the effects of various treatment strategies against Botrytis in the vineyard and demonstrates the importance of the prophylactic method.

Aboveground and belowground ectomycorrhizal (ECM) communities associated with different age classes of the exotic plantation species Pinus radiata were investigated over the course of two years in the North Island of New Zealand. ECM species were identified with a combined approach of morphological and molecular (restriction fragment length polymorphism (RFLP) and DNA sequencing) analysis. ECM species richness and diversity of a nursery in Rotorua, and stands of different ages (1, 2, 8, 15 and 26 yrs of age at time of final assessment) in Kaingaroa Forest, were assessed above- and belowground; furthermore, the correlation between the above- and belowground ECM communities was assessed. It was found that the overall and stand specific species richness and diversity of ECM fungi associated with the exotic host tree in New Zealand were low compared to similar forests in the Northern Hemisphere but similar to other exotic plantations in the Southern Hemisphere. Over the course of this study, 18 ECM species were observed aboveground and 19 ECM species belowground. With the aid of molecular analysis the identities of Laccaria proxima and Inocybe sindonia were clarified. In the aboveground study, five species were found associated with P. radiata that were previously not reported with this host in New Zealand (Inocybe sindonia, Lactarius rufus, Lycoperdon gunii, Rhizopogon pseudoroseolus and Wilcoxina mikolae). Belowground, the species Psudotomentella sp., P. tristis, R. luteorubescens, Tomentella sp., Wilcoxina mikolae were found as new associates of P. radiata in New Zealand, additionally nine ECM types were found that could not be identified with molecular analysis. There was little correlation between the species fruiting and the species colonising root tips. Only seven species were found in common between the above- and belowground communities, furthermore the dominant species aboveground were not observed in the belowground ECM communities. The influence of host age on the above- and belowground ECM communities of different age classes of P. radiata plantations was investigated. The aboveground species richness increased from the nursery to the oldest age group investigated (26 yrs), while diversity increased to the 15 yr old age group and decreased slightly to the oldest stand. A clear sequence of ECM species changes was observed to be related to stand age with a growing complexity over the chronosequence. The belowground ECM communities showed a different picture and richness and diversity initially decreased from the nursery to the outplanting but increased thereafter. Belowground no change in ECM composition that was directly related to the age of the host was observed, but two distinct groups of ECM species were found – a 'young' and a 'plantation forest' group, with the respective discriminating species being Rhizopogon rubescens and Type unknown Basidiomycete/Amanita muscaria. Another aspect of the study was the fate of the nursery ECM species in the outplanting and the arrival of non-nursery species. The ECM communities of seedlings in the nursery were investigated in 2006 and these seedlings were followed up over eight assessments in the field for one year, furthermore data from the 1-, 2 and 8 yr old plantation stands was analysed. It was found that the nursery species do survive the first year of outplanting and are dominant in the first year. The first non-nursery species occurred six months after outplanting but was only in minor abundance. Nursery ECM were dominant for two years after the seedlings were planted, and were completely replaced after seven years. Rhizopogon rubescens was found to be the most persistent and dominant species in the outplanting, facilitating the successful establishment of the seedlings in the plantation forest.