Digital Image Analysis of Internal Light Spots of Appressoria of Colletotrichum acutatum

ABSTRACT The initial penetration process of appressoria of Colletotrichum acutatum on almond leaves was studied using digital image analysis of light micrographs and scanning electron microscopy. For image analysis, a series of sequential, partially focused digital micrographs of appressoria was analyzed to generate a single, completely focused montage image with a continuous in-focus depth of field. In studies on the development of the internal light spot (ILS), we observed that 50.4% of the appressoria formed an ILS after leaves were inoculated and incubated for 12 h at 20 degrees C, and that this increased to 95.8% after 24 h. Comparative image analyses of appressoria with and without ILSs using depth relief mapping and line profile software options showed that the ILS had a depth relief that was below that of the leaf surface. Depth relief analysis in the ILS region during incubation revealed an increase in depth in this area of up to 1.8 mum in some of the appressoria. A comparative morphological study of the ILS in montage images and the penetration pore of appressoria in scanning electron micrographs showed similar shapes and dimensions of the two structures in the appressorium. Light micrographs of histological sections confirmed fungal penetration and internal vesicle formation in almond leaves within 36 h after inoculation and incubation at 20 degrees C. This study represents the first direct evidence that the ILS in appressoria corresponds to the penetration pore and the developing penetration peg using a rapid, digital image analysis technique.     

Essential oils content and antioxidant properties of peel ethanol extract in 18 lemon cultivars

Composition of essential oils and antioxidant activity of peel ethanol extract were analyzed in 18 local lemon cultivars. Essential oils composition was determined by GC/FID analysis, and antioxidant activity with the ABTS method. Fruit weight, polar and equatorial diameters, peel thickness, seed number, juice percentage, titratable acidity and juice pH were also determined for each cultivar. The main component in the peel essential oil was limonene, accounting for 72.5–76.4%, followed by β-pinene (11.6–18.7%). Several other monoterpene hydrocarbons were also identified at appreciable contents, namely terpinene (2.88–8.26%), α-pinene (1.4–1.5%) and myrcene (0.95–1.12%). No clear relationship could be shown between the antioxidant activity and the essential oil composition of the extracts. In this study, cultivars with higher essential oil content and antioxidant activity were identified. Data were subjected to analysis of variance using ANOVA and means were compared by the Duncan test.     

A Greenhouse Test for Screening Sugar Beet (Beta Vulgaris) for Resistance to Rhizoctonia Solani

Rhizoctonia solani Kühn is a serious plant pathogenic fungus, causing various types of damage to sugar beet (Beta vulgaris L.). In Europe, the disease is spreading and becoming a threat for the growing of this crop. Plant resistance seems to be the most practical and economical way to control the disease. Experiments were carried out to optimise a greenhouse procedure to screen plants of sugar beet for resistance to R. solani. In the first experiment, two susceptible accessions were evaluated for root and leaf symptoms, after being grown in seven different soil mixtures and inoculated with R. solani. The fungus infected all plants. It was concluded that leaf symptoms were not reliable for the rating of disease severity. Statistically significant differences between the soil mixtures were observed, and there were no significant differences between the two accessions. The two soil mixtures, showing the most severe disease symptoms, were selected for a second experiment, including both resistant and susceptible accessions. As in the first experiment, root symptoms were recorded using a 1–7 scale, and a significant expression of resistance was observed. The average severity of the disease in the greenhouse experiment generally was comparable with the infection in field experiments, and the ranking of the accessions was the same in the two types of experiments. It was concluded that evaluation procedures in the greenhouse could be used as a rapid assay to screen sugar beet plants for resistance to R. solani.     

A viability study of Fallopia japonica stem tissue

Vegetative regeneration of intact and split nodes of Fallopia japonica (Houtt.) Ronse Decraene was the focus of this greenhouse experiment. Stem material, collected in May, July and September 1997, was cut into 40-mm-long sections and incubated on the surface of moist compost in a greenhouse. All samples showed some regrowth of buds and new shoots from both intact and split nodes in any season. A maximum number of 0.25 buds per intact node was recorded after 6 days in a spring experiment. The vegetative regrowth of shoots generally appeared after 1 week and increased over time with a maximum mean height of 77 mm after 27 days and a maximum number of 0.39 shoots per intact node after 13 days. Adventitious roots developed from both intact and split nodes during both summer and autumn experiments. From this study, a maximum of 90 stem tissue propagules per square metre of F. japonica stand has been estimated. If the plant is cut as part of a control programme, these propagules can produce shoots with growth rates of up to 2.9 mm per day with a good chance of the development of adventitious roots. This considerable potential for vegetative regeneration of stem tissue shows a clear need for guidelines on the safe management and disposal of F. japonica plant material.     

Bacterial populations associated with rice seed in the tropical environment

ABSTRACT During the 1995 wet season, harvested rice seed was collected from farmers' fields at different locations in Iloilo, Philippines. Bacterial isolations from crushed seed yielded 428 isolates. The isolates were characterized by BOX-polymerase chain reaction fingerprinting of total genomic DNA and represented 151 fingerprint types (FPT). Most FPTs were found on a single occasion, although matching fingerprints for isolates from different samples also were found. Identifications were made by cellular fatty acid methyl ester analysis and additional use of Biolog GN/GP MicroPlates and API 20E/50CHE systems. The predominant bacteria were Enterobacteriaceae (25%), Bacillus spp. (22%), and Pseu-domonas spp. (14%). Other bacteria regularly present were identified as Xanthomonas spp., Cellulomonas flavigena, and Clavibacter michiganense. Of the total number of isolated bacteria, 4% exhibited in vitro antifungal activity against Rhizoctonia solani or Pyricularia grisea. Two percent of isolates were pathogens identified as Burkholderia glumae and Burkholderia gladioli. Five percent of isolates induced sheath necrosis on only 50 to 90% of inoculated plants and were related to Bacillus pumilus, Paenibacillus spp., Pseudomonas spp., and Pantoea spp.     

Molecular characterization of <Emphasis Type="Italic">Xiphinema brevicollum</Emphasis> (Nematoda: Longidoridae) from the Czech Republic

Populations of Xiphinema brevicollum occurring in the Czech Republic were described morphologically and molecularly. Published species-specific primer set BL18 and BV3 was used to amplify three populations of X. brevicollum from the Czech Republic. These primers were tested against 9 species of Xiphinema and 11 species of Longidorus. Amplification was also observed for X. inaequale, X. italiae and X. lambertii. Three additional markers, mitochondrial cytochrome c oxidase subunit 1, ribosomal D2/D3 expansion segment of the 28S gene and 18S gene, were amplified and sequenced for X. brevicollum, X. inaequale and X. lambertii belonging to X. americanum-group. Comparison of cox1 sequences of X. brevicollum from the Czech Republic with X. taylori from the Slovak Republic (accession number AM086702) suggested that these populations represent the same species.     

Branch cankers on citrus trees in Spain caused by Phytophthora citrophthora

Considerable losses of citrus trees have been observed in the major citrus-growing areas of Spain. Samples were collected from 132 orchards, and isolations and pathogencity tests were conducted to determine the aetiology of a serious canker disease. Affected trees showed cankers on the scion that frequently began on the branches. Three Phytophthora species were identified based on their morphological, cultural, physiological and molecular profiles. Phytophthora citrophthora was the main species associated with this new syndrome in 114 orchards. Phytophthora nicotianae (syn. P. parasitica ) was isolated from nine orchards as the sole Phytophthora species and in coinfection with P. citrophthora from another nine orchards. Phytophthora citricola was isolated only from one orchard. In stem-inoculation studies conducted under greenhouse conditions, clementine mandarin cv. Hernandina and sweet orange cv. Navel Late were more susceptible to P. citrophthora than sour orange and Carrizo citrange rootstocks. Clementine cv. Hernandina was also highly susceptible in field inoculation experiments. In agreement with field surveys, clementine mandarin cultivars were the most affected, their rootstocks remaining healthy. Phytophthora citrophthora was found to be the predominant species in orchard soils; however, P. nicotianae was also isolated. This information changes the scenario of diseases caused by Phytophthora spp. in Spain and consequently, the present knowledge of epidemiology and the effectiveness of the current control measures should be reassessed.     

Relationship between the incidence of latent infections caused by <Emphasis Type="Italic">Monilinia</Emphasis> spp<Emphasis Type="Italic">.</Emphasis> and the incidence of brown rot of peach fruit: factors affecting latent infection

Five field experiments were performed in commercial orchards located in Lleida (Spain) over three growing seasons, 2000–2002, in order to estimate the relationship between the incidence of latent infection caused by Monilinia spp. in peaches and the incidence of post-harvest brown rot. No latent infection was recorded at popcorn and the maximum incidence occurred pre-harvest; in some orchards a second peak was detected during the pit hardening period. Monilinia laxa is the most prevalent species isolated from peaches with brown rot. There was a positive correlation between the incidence of latent infection and that of post-harvest brown rot. The average incidence of latent infection during the crop season explained 55% of the total variation in the incidence of post-harvest brown rot. The effect of temperature (T) and duration of wetness (W) on the incidence of latent infection in peach and nectarine orchards was analysed using multiple regression. The regression analysis indicated that T and W jointly explained 83% of the total variation in the incidence of latent infection. The model predicts no latent infections when T < 8°C, and >22 h wetness are required when T = 8°C but only 5 h at 25°C are necessary for latent infection to occur. The incidence of brown rot and latent infection of peaches caused by M. laxa under controlled experimental conditions were also affected by T and W, as well as by fruit maturity and inoculum concentration. Latent infections were produced in fruit when T was not suitable for the development of brown rot symptoms. In these experiments more than 4–5 h of daily wetness were required after embryo growth in fruit sprayed to run-off with an inoculum concentration higher than 10⁴ conidia ml⁻¹ of M. laxa for brown rot and latent infections to develop. The fitted model obtained from the field data was able to predict the observed data obtained under controlled environmental conditions.     

Implementation of an artificial reaction control in a TaqMan method for PCR detection of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> race 3 biovar 2

A previously published TaqMan PCR test for R. solanacearum race 3 biovar 2 was modified to enable both the validation of negative results and the confirmation of positive results in a closed-tube system. Negative results were validated through the use of a reaction control plasmid, designated pRB2C2, which was designed to generate a 94bp product using the same amplimers targeting the primary diagnostic 68bp sequence in R. solanacearum race 3 biovar 2 DNA. SYBR Green was included in the reaction mix to facilitate the identification of post-reaction products using melt peak analysis. The 94bp reaction control had a melt peak temperature of about 90°C, while the diagnostic target amplicon had a melt peak temperature of about 83°C; thus positive results could be easily confirmed and distinguished from the reaction control product. Addition of pRB2C2 at 100 copies per reaction had no effect on the sensitivity of the TaqMan assay for R. solanacearum race 3 biovar 2, and the modified assay successfully detected R. solanacearum race 3 biovar 2 in infected, asymptomatic tomato stems and leaves as well as in potato tubers and stems.