Use of real-time PCR to examine the relationship between disease severity in pea and <Emphasis Type="Italic">Aphanomyces euteiches</Emphasis> DNA content in roots

Aphanomyces euteiches causes severe root rot of peas. Resistance is limited in commercial pea cultivars. Real-time fluorescent PCR assay specific for A. euteiches was used to study the relationship between disease severity and pathogen DNA content in infected peas. Five pea genotypes ranging in levels of resistance were inoculated with five isolates of A. euteiches. Plants were visually rated for disease development and the amount of pathogen DNA in roots was determined using the PCR assay. The susceptible genotypes Genie, DSP and Bolero tended to have significantly more disease and more pathogen DNA than the resistant genotypes 90-2079 and PI 180693. PI 180693 consistently had less disease, while 90-2079 had the lowest amount of pathogen DNA. The Spearman correlation between pathogen DNA quantity and disease development was positive and significant (P < 0.05) for three isolates, but was not significant for two other isolates. This suggests that the real-time PCR assay may have limited application as a selection tool for resistance in pea to A. euteiches. Its utility as a selection tool would be dependent on the correlation between disease development and pathogen DNA content for a given pathogen isolate. The accuracy and specificity of the real-time PCR assay suggests considerable application for the assay in the study of mechanisms of disease resistance and the study of microbial population dynamics in plants.     

Biological Control of Sclerotinia Diseases of Rapeseed by Aerial Applications of the Mycoparasite Coniothyrium minitans

Indoor and field experiments were conducted to evaluate the efficacy of applying the mycoparasite Coniothyrium minitans to the aerial parts of rapeseed plants at the flowering stage to control sclerotinia diseases caused by Sclerotinia sclerotiorum. Under controlled conditions, a petal inoculation technique was used to determine the effect of conidial suspensions of C. minitans on suppression of sclerotinia leaf blight. Results showed that C. minitans was effective in inhibiting infection initiated by ascospores of S. sclerotiorum on flower petals by restricting mycelial growth of the pathogen. Suppression of lesion development was related to the conidial concentration of C. minitans, with larger lesions at low concentration (5×10³conidia ml⁻¹), but smaller lesions at high concentration (5×10⁴ conidia ml⁻¹ or higher). When C. minitans-treated rapeseed leaves were inoculated with mycelia of S. sclerotiorum, C. minitans failed to prevent infection of leaves, but caused a significant reduction in number of sclerotia produced on the diseased leaves. No significant difference in efficacy was detected between the two isolates of C. minitans, LRC 2137 and Chy-1, on the two rapeseed cultivars, Westar (spring type) and Zhongyou 821 (winter type). Results of field trials showed a significant reduction of stem rot of rapeseed in four (1997, 1999, 2003 and 2004) out of five years by aerial application of C. minitans, compared with controls. No significant difference in suppressive efficacy was observed between the treatments of C. minitans (10⁶ conidia ml⁻¹), C. minitans (10⁶ conidia ml⁻¹) + benomyl (50 μg ml⁻¹) and benomyl (100 μg ml⁻¹) in 2003, and between the treatments of C. minitans (10⁶ conidia ml⁻¹), C. minitans (10⁶ conidia ml⁻¹) + vinclozolin (100 μg ml⁻¹) and vinclozolin (500 μg ml⁻¹) in 2004. Sclerotia of S. sclerotiorum collected from diseased plants in plots treated with C. minitans in 1999, 2000 and 2003, or with C. minitans + benomyl in 2003 were infected by C. minitans at frequencies ranging from 21.3 to 54.5%. This study concludes that aerial spraying of C. minitans is an effective method for controlling sclerotinia diseases of rapeseed.     

Implications of on-farm research for local knowledge related to fruit flies and the weaver ant Oecophylla longinoda in mango production

We interviewed half of the mango-growers in northern Benin, including 15 farmers involved in a regional fruit fly project, and held focus group discussions with women fruit-pickers. They were asked about pest management and their knowledge of a weaver ant, Oecophylla longinoda. All considered low yields due to fruit flies to be the principal constraint upon mango production, estimating economic losses to be between 20 and 45%. None could recognize damage during the first 2 days after fruit fly egg deposition. On-farm research persuaded farmers to stop using insecticides and it also changed negative perceptions of Oecophylla. Over 80% of the farmers involved in on-farm research, compared to 25% of those not involved, reported Oecophylla to be beneficial. All fruit-pickers knew that ants protected mango from fruit flies, with 60% attributing better mango quality in terms of appearance, shelf-life and sweetness to the presence of Oecophylla. Nevertheless, 40% of the pickers still considered weaver ants a nuisance pest during harvest. Ways of reducing this nuisance need to be developed for Oecophylla to gain wider acceptance by mango-growers.     

Development of specific PCR primers for identification and detection of <Emphasis Type="Italic">Phytophthora capsici</Emphasis> Leon

A PCR-based method was developed for the identification and detection of Phytophthora capsici in pepper plants. Three PCR primers (CAPFW, CAPRV1 and CAPRV2) specific for P. capsiciwere designed based on the sequence of its internal transcribed spacer regions. CAPFW/CAPRV1 amplify a 452 bp product from P. capsici DNA whereas CAPFW/CAPRV2 a 595 bp fragment; neither set amplifies DNA from pepper or several fungi pathogenic to pepper. In conventional (single-round) PCR, the limit of detection was 5 pg DNA for both primer sets, whereas in nested PCR the detection limit for both was of 0.5 fg. However, when the dilution series of target DNA were spiked with plant DNA, amplification declined two-fold in both conventional and nested PCR. The CAPFW/CAPRV2 set in conventional PCR was used to detect P. capsici DNA in inoculated plants. Detection occurred as soon as 8h post-inoculation in stem samples from infected but still symptomless plants. The method was also tested to detect fungal DNA in infected soils.     

Reaction of maize,sorghum and Johnson grass to Peronosclerospora sorghi

Development of sorghum downy mildew, incited by Peronoscleospora sorghi (Weston and Uppal) C.G. Shaw, on maize, sorghum and Johnson grass was investigated at two locations in Uganda during three seasons (1994 and 1995). More sorghum downy mildew developed on the Johnson grass and sorghum than on the maize at all locations and in all seasons. No significant differences were observed in sporulation of P. soghi on the three hosts. Leaf shredding occurred on the three hosts but was the least on maize. Cross-inoculation with both conidia and oospores was achieved on the three hosts. Since the fungal population formed oospores and sporulated readily on the three hosts, which is typical of the sorghum strain, the disease in Uganda is attributed to the sorghum strain.     

Variable response of open-pollinated seedling progeny of avocado to Phytophthora root rot

Phytophthora root rot, caused byPhytophthora cinnamomi Rands, is the most important disease of avocado (Persea americana Miller). In an attempt to identify root rot-resistant rootstocks that could ultimately be used under conditions in southern Florida, we screened open-pollinated progeny of avocado from the National Germplasm Repository in Miami. From 1996 to 1998, a total of 2,355 seedlings from 51 accessions were examined in potting mix artificially infested withP. cinnamomi. Most seedlings developed severe root rot, but tolerance was observed in some families (i.e., progeny of certain accessions). Although the most susceptible families developed mean disease ratings of up to 97% root necrosis, mean ratings for the most tolerant families were less than 60%. There was also a strong relationship between the racial background of the female parent and the tolerance of seedlings. Seedlings of the West Indian race and hybrids between it and the Guatemalan race were significantly more tolerant than those from other parents (P< 0.05). Individuals in several families developed < 50% root necrosis, the arbitrary standard of tolerance in this study. Twelve families accounted for 82% (188 of 229) of the tolerant seedlings, and only two of these did not have a West Indian or Guatemalan × West Indian pedigree. Broad-sense heritability for PRR tolerance was 0.45. This is the first report on the inheritance of PRR tolerance in avocado and on the influence of genotype and racial pedigree under controlled conditions.     

Deep‐sequencing revealed Citrus bark cracking viroid (CBCVd) as a highly aggressive pathogen on hop

Newly emerging or re‐emerging diseases are a constant and significant threat to agricultural production, so prompt and accurate identification of the causative agents is required for rapid and appropriate disease management. Classical methods of pathogen detection can be successfully supplemented by next‐generation sequencing (NGS), whereby sequence analysis can help in the discovery of new or emerging diseases. In 2007, hop growers in Slovenia reported the appearance of severely stunted hop plants, a phenomenon that spread rapidly within hop gardens and among farms. Classical diagnostic methods were unable to detect a new pathogen; therefore, single step high‐throughput parallel sequencing of total RNA and small RNAs from plants with and without symptoms was employed to identify a novel pathogen. The sequences were assembled de novo and also mapped to reference genomes, resulting in identification of a novel sequence of Citrus bark cracking viroid (CBCVd) in the stunted hop plants. Furthermore, the presence of this novel pathogen on hop was confirmed by RT‐PCR analysis of 59 plants with symptoms from 15 hop gardens, representing the main outbreak locations identified by systematic disease monitoring, and small RNA Illumina sequencing of the bulked RNA sample. The high infectivity of the newly identified CBCVd was also confirmed by biolistic inoculation of two hop cultivars, which developed aggressive symptoms in controlled conditions. This study shows the feasibility of deep sequencing for the identification of causative agents of new diseases in hop and other plants.     

Genetic diversity of Pueraria lobata (kudzu) and closely related taxa as revealed by inter-simple sequence repeat analysis

Kudzu (Pueraria lobata) is a noxious weed infesting some areas of the USA. Knowledge of its genetic variation in both native and invasive areas can lead to effective biological control measures. Inter-simple sequence repeat (ISSR) variations were studied in P. lobata and its four closely related congeneric species (P. edulis, P. montana, P. phaseoloides and P. thomsoni). ISSR results allowed a clear separation of these five species. For P. lobata, 108 plants from China and USA were analysed. The samples from the US were genetically closer to Chinese P. lobata populations than to other congeneric populations. High genetic differentiation was found for P. lobata, P. montana and P. thomsoni in Chinese samples. High genetic diversity and low population differentiation was found in P. lobata samples of the US. This supports the hypothesis of multiple introductions into the USA from different sources in Japan or China, followed by subsequent gene exchange and recombination.     

A dynamic model simulating infection of apple leaves by Venturia inaequalis

A new dynamic model of the infection of apple leaves by Venturia inaequalis is described. The model begins with the release of spores by rain and incorporates the effect of light on the discharge of ascospores from pseudothecia. The model then simulates infection through the sub-processes of germination, appressorium formation and penetration, separately for ascospores and conidia landed concurrently on wet leaves. The rate of the infection process is determined using different equations for ascospores and conidia. Spore mortality when leaves dry is determined by the stage of infection and RH in the dry period. The infection process is driven by surface wetness, temperature and RH. The progress of each infection period is measured as infection efficiency (IE), namely the percentage of landed spores which have penetrated and thereby infected leaves. The final IE quantifies the favourability of weather in each infection period. In orchard tests in each of three years, the new model detected crucial infection periods in spring and early summer which accounted for outbreaks of leaf scab. These periods were not detected by a static model based on Mills'criteria. The models performed similarly in detecting infection periods later in summer.     

Storage performance of clementines treated with hot water,sodium carbonate and sodium bicarbonate dips

Summary

The potential of using hot water (2.5 min at 45°C), 2% sodium carbonate or 2% sodium bicarbonate solutions alone or combined with hot water, for control of Penicillium digitatum (green mould) was investigated on commecially ripe clementines. The effect of such treatments on the post-harvest storage life of clementines was determined during two months analysing weight loss, increase in rind deformation and internal maturity. Carbonate and bicarbonate solutions effectively control green mould during storage but hot water does not. Compared with the control and hot-water treated fruits, clementines treated with carbonate and, to a lesser extend with bicarbonate, showed higher weight loss and lower firmness during storage. In contrast, lower increase in ripening index was found when the fruits were treated with carbonate or bicarbonate. Fruits treated with hot water alone or combined with carbonate exhibited no change in ripening during the first month of storage. Rinsing the fruit significantly reduces weight and firmness loss in the fruits treated with carbonate and bicarbonate, but did not change the ripening index. The primary finding of this work was that carbonate and bicarbonate treatments might significantly increase the loss in weight and firmness during storage. Rinsing the fruit immediately after treatment appeared to be a simple solution to avoid these losses in quality.