Characterization of Botrytis cinerea Isolates Present in Thompson Seedless Table Grapes in the Central Valley of Chile.

Botrytis cinerea isolates from flowers and berries of Vitis vinifera 'Thompson seedless' (grapevine) were characterized in terms of two transposable elements (TEs) Boty and Flipper, random amplified polymorphic DNA (RAPD), infection levels, and resistance to iprodione. The isolates were collected from grapevines under fungicide programs of variable numbers of iprodione applications, and replicated in three Chilean Central Valley locations. Recovery was repeated from clusters collected at four phenological stages. Highest infection levels were found at bloom. Fungicide programs including one iprodione application or a combination of other fungicides were most effective for reducing gray mold symptoms. A total of 457 isolates collected from fungicide programs including only one iprodione application, and the control program, were tested for the presence of TEs. In all locations and during all phenological stages, transposa isolates (containing both TEs) were most common, followed by Boty. Vacuma isolates (containing neither TE) were identified at very low levels in two locations and only in the control treatment, and isolates with only Flipper were not detected at any time or location. Vacuma and Boty isolates were all sensitive to iprodione, while transposa isolates showed a wide range of resistance. Based on response to iprodione, the presence of TEs, and presence of vegetative-incompatibility alleles (Bc-hch), the isolates studied belong to B. cinerea Group II, a phylogenetic species within B. cinerea. Hierarchical analysis of molecular variance and genetic diversity analyses of the RAPD genotypes showed a genetic differentiation linked to location, but it was not related to geographic distance. Moreover, a genetic differentiation related to the phenological stage of grapes was also detected.

Photosynthetic metabolism during phosphate limitation in a legume from the Mediterranean-type Fynbos ecosystem.

Phosphorus (P) is an essential mineral, required for crucial plant genetic, metabolic and signaling functions. Under P deficiency, normal physiological function can be disrupted, especially photosynthetic metabolism. The majority of photosynthetic studies of P stress has been on model organisms, and very little is known about plants that evolved on P deficient soils. Aspalathus linearis (Burm.f.) R.Dahlgren, a native to the Mediterranean ecosystem of South Africa was used to study the photosynthetic responses during short-term P limitation. A. linearis seedlings were cultured under glasshouse conditions and exposed to short-term P stress. Leaf photosynthetic gas exchange was coupled with metabolic analyses. In spite of the decline in leaf cellular Pi, the photosynthetic rates remained unchanged. These leaves also maintained their levels of light harvesting and reaction center pigments. The efficiency of the light reactions' utilization of ATP and NADPH increased during P-stress. Leaf glucose levels decreased during P-stress, while sucrose concentrations remained unaffected. These results show that during short-term P-stress, A. linearis can maintain its photosynthetic rates by altering the structural and functional components of the light reactions.

Molecular identification of Botrytis cinerea, Botrytis paeoniae and Botrytis pseudocinerea associated with gray mould disease in peonies (Paeonia lactiflora Pall.) in Southern Chile.

BACKGROUND: In Chile, the peony is the most important ornamental flower exported from the country. Gray mould is a phytopathological problem of this crop. This disease is caused by Botrytis cinerea and Botrytis paeoniae. AIMS: We carried out the first survey of Botrytis species associated with peony gray mould in Southern Chile to estimate the diversity of these pathogens. METHODS: Diseased peony leaves were collected from seven locations in Southern Chile covering a distance of 300km. The Botrytis isolates obtained were studied by morphological and molecular methods. Finally, a PCR assay using primers based on the necrosis and ethylene-inducing protein gene (nep1) was used to specifically identify B. paeoniae. RESULTS: Seventeen isolates belonging to Botrytis genus were obtained, and all of them were pathogenic to peonies when inoculated in plants grown in a greenhouse. Morphological analyses showed that four isolates shared common characteristics, which distinguish them from the rest. Homology and phylogenetic analysis of G3PDH, as well as determination of the Bc-hch allele, allowed us to identify 12 isolates as B. cinerea, 4 as B. paeoniae and one isolate as Botrytis pseudocinerea. The PCR assay was found to be specific to B. paeoniae, amplifying a single band of 470bp. CONCLUSIONS: Three Botrytis species involved in peony gray mould disease are present in Chile. This is the first time that both B. paeoniae and B. pseudocinerea have been reported to be present in the country and also that they affect peonies. Finally, to our knowledge, the PCR based method herein described is the first of its kind to be used to identify B. paeoniae.

Differential gene expression in proteoid root clusters of white lupin (Lupinus albus).

Proteoid root clusters are induced by P deficiency in white lupin. In their mature stage, these roots excrete organic acids (mainly citrate), thus allowing this species to acquire P from sparingly soluble sources. To screen for P-regulated genes expressed during the period of citrate efflux, an experimental model based on proteoid root clusters contrasting in citrate efflux was developed. The feasibility of this model in identifying differential gene expression was assessed over a population of mRNAs from P-starved and P-starved rescued proteoid root clusters, sampled 24 and 72 h after P addition to 24 days P-starved white lupin. Approximately 1500 bands of cDNA were displayed by differential display of 21-primer pair's combination; 52 differentially expressed bands, either up- or down-regulated after P addition, were observed. Sequence analysis of 17 of them revealed that they represent distinct cDNAs. A subsample of seven cDNAs was analysed by northern-blot, showing that six were truly differential products. Transcripts coding for enzymes involved in carbon flux (glyceraldehyde 3-phosphate dehydrogenase), glycolytic bypass (phosphoenolpyruvate carboxylase), Pi recycling (sulpholipid synthase), and two unknown cDNAs were shown to be down-regulated by P supply. Besides, an up-regulated transcript coding for a putative auxin-induced protein was identified, whereas P addition did not significantly affect expression of a transcript for cyclophilins. These results show the feasibility of using P-starved and P-starved rescued proteoid root clusters as an experimental model to detect and examine the molecular changes occurring in root clusters during the period of citrate efflux in white lupin.

Characterization, In Vitro Culture, and Molecular Analysis of Thecaphora solani, the Causal Agent of Potato Smut.

ABSTRACT The fungus Thecaphora solani (syn.: Angiosorus solani), the causal agent of potato smut, was cultivated in vitro for the first time. Teliospores obtained from galls of infected potato plants were used to inoculate commonly used solid and liquid media. The teliospores produced two kinds of vegetative tissue depending on the nutrient status of the media. A very slow radial-growing, hyaline, and septate mycelium, as usually seen in most of the in vitro-cultivated filamentous fungi, was obtained in wateragar medium after 30 to 40 days. On the other hand, a white, sponge-like mycelial mass was obtained in HCM + 1% activated charcoal, and on common potato dextrose agar or malt-yeast-peptone solid or liquid media, after 40 to 50 days under lab conditions. The identity among teliospores and the sponge-like mycelial mass was corroborated by DNA fingerprinting and partial sequencing of the large subunit (LSU) rDNA region. The sexual cycle of the pathogen was completed under lab conditions based on the development of teliospores on the sponge-like mycelial mass. The first attempt to reproduce the disease under controlled conditions was successful, inducing a gall in a cv. Desirée potato explant cultivated in vitro inoculated with radial-growing mycelia. Phylogenetic analysis of LSU rDNA data of the genus Thecaphora and other smut fungi confirmed the initial classification of the pathogen as T. solani.

Phosphate deficiency regulates phosphoenolpyruvate carboxylase expression in proteoid root clusters of white lupin.

Proteoid roots play a major role in enabling white lupin (Lupinus albus L.) to adapt to phosphate (Pi) deficiency. Such roots release citrate from proteoid rootlets, which allows this species to mobilize Pi from sparingly soluble Pi sources. Release of citrate is preceded by a significant accumulation of organic acids, in which a Pi deficiency-inducible phosphoenolpyruvate carboxylase (PEPC) activity has been involved. To gain an insight into this adaptive mechanism, the expression of three different transcripts coding for PEPC was examined in proteoid rootlets of Pi-starved and Pi-starved-and-rescued white lupin. Semi-quantitative reverse transcriptase (RT)-PCR experiments performed with gene-specific primers targeted to the 3'-end region of the corresponding cDNAs revealed that the transcripts for these three PEPCs differentially accumulate in both Pi-starved and Pi-starved-and-rescued proteoid rootlets. Semi-quantitative RT-PCR analysis in Pi-starved proteoid rootlets sampled at different times after being rescued from Pi deficiency showed that Pi levels differentially down-regulated the three PEPC transcripts. RT-PCR experiments were further extended to Pi-starved and Pi-fed whole roots, cotyledons, and leaves on which a tissue-specific, Pi-dependent PEPC expression was observed. These results indicate that there exists at least three different transcripts coding for PEPC in proteoid root clusters of white lupin, whose expression are differentially regulated by Pi.