Occurrence of Phaeomoniella chlamydospora on grapevine planting material in Sardinia and its control with combined hot water and cyproconazole treatment.

The occurrence of Phaeomoniella chlamydospora was investigated during the propagation process of an Italian nursery, and combined hot water and cyproconazole treatments were carried out to limit its spread in nursery plants. In the three–year period 2005–07, cutting and graft samples (scion cv Sangiovese, rootstock cvs 140Ru in 2005 and 1103P in 2006–07) were taken during the propagation process at several infection risk stages in order to assess the presence of P. chlamydospora. Moreover, in 2005 and 2006 cuttings from esca–symptomatic grapevines (scion cv Sauvignon blanc, rootstock cvs 140Ru in 2005 and 1103P in 2006) were treated at different stages of the propagation process. In 2007, artificially infected 1103P cuttings were treated after inoculation with P. chlamydospora (107 conidia ml-1). Cuttings and callused graftlings were treated by dipping in a hot water bath at 50°C for 30 min (HWT) or in a cyproconazole suspension (0,1 g a.i. l-1) for at least 12 h, in different combinations. At each stage of infection risk and at each treatment woody material was collected and grown in pot, avoiding accidental contamination, or in the field nursery for one season and then destructively examined. DNA was extracted from wood collected at different points along the plant and analysed by nested PCR with Pch-specific primers. One-mm thick woody slices from artificially inoculated cuttings were plated on MEA plus antibiotics and fungicides. Despite the extended wood discoloration, P. chlamydospora occurrence on nursery plants was scarce in the three–year period. Planting material contamination may have resulted from infected mother plants (from 0.0 to 6.7 % of infected cuttings were obtained directly from non-symptomatic mother plants) or from the propagation process, particularly during the stages following grafting (0.0 to 23.3 % of infected grafts). Canes from esca–diseased mother plants were always contaminated, but in quite different percentages (about 30 % of the cuttings examined in 2005, from 1.9 to 4.1 % in 2006 and 2007). However, no final conclusion could be drawn about which stages played a major role in the contamination of nursery plants, due to the low and irregular infection frequencies detected in the propagation process. As regards P. chlamydospora control, HWT performed on cuttings before or after cold storage influenced vegetative growth depending on both the cultivar and the growth conditions, but it was deleterious on callused graftlings. Natural contamination on nursery material in 2005 and 2006 was insufficient to assess the effectiveness of treatments. In 2007, HWT and cyproconzole treatments alone were not effective in reducing the percentage of infection in cuttings that were artificially inoculated. Only cyproconazole immediately followed by HWT significantly reduced the number of infected cuttings, but it was not sufficient to eradicate the pathogen

Expression of grapevine leaf stripe disease foliar symptoms in four cultivars in relation to grapevine phenology and climatic conditions

Grapevine leaf stripe disease (GLSD) symptom expression was analysed in four vineyards and four cultivars, in Sardinia (Italy), taking into account ten-year annual and five-year monthly surveys. The cumulative incidence of symptomatic plants reached high values on Sauvignon blanc, Cabernet sauvignon and Cannonau (81.9, 79.4 and 66.5% respectively), but low on Merlot (25.1%). Symptoms appeared during or before the 50% flowering stage and maximum increments were assessed in June and partially in July. Annual incidence of foliar symptoms fluctuated in the ten years of the survey. Positive regressions were found between incidence of vines that exhibited foliar symptoms in year n but were symptomless in year n-1 and rain parameters in the 30 days after stabilization of mean temperature around 10°C, when colonization of pruning wounds begins. This relationship could suggest the involvement of new infections or re-infections on symptom expression in the following growth season. Significant regressions between incidence of vines that exhibited foliar symptoms in year n but were symptomless in the year n+1 and climatic parameters were also recorded. High temperatures and low rainfall in the period from pre-flowering to veraìson were conducive to a higher number of asymptomatic plants. Regarding monthly foliar symptoms evolution, an increase in temperature from 50% sprouting until June led to a greater number of new symptomatic plants. On the other hand, a smaller percentage of new symptomatic plants was associated with an increase in temperature from June to July, which may have influenced vine water balance and transport of toxins by the sap flow

Identification and characterization of Burkholderia isolates obtained from bacterial rot of saffron (Crocus sativus L.) grown in Italy

Twenty five isolates of Burkholderia gladioli, the causal agent of a bacterial disease recently reported on saffron (Crocus sativus L.) grown in central Sardinia (Italy), were characterized using different approaches. The characteristic symptoms of the disease on saffron plants were rot of emerging shoots and leaves and spots on leaves and corms. In the field, the disease was destructive and reduced flowering by about 80%. Two types of colonies of bacteria cultured from affected plants were selected on the basis of their characteristic morphology and pigment production on nutrient-glucose-agar. One type was round, wrinkled, and producing yellowish pigment; while the second was round, smooth and without pigment. All 25 selected isolates were pathogenic on saffron leaves and corms. Ten were pathogenic on gladiolus and lily leaves. None of the tested isolates was pathogenic on onion plants. The isolates were characterized by conventional tests, Biolog, PCR and PCR-RFLP analysis. Conventional tests and PCR identified all isolates as B. gladioli. PCR-RFLP analysis of 16S rDNA products digested with the three restriction enzymes Alu I, Dde I and Bss KI, identified ten of the isolates as B. gladioli pv. gladioli. Sequencing and comparison of the 16S rDNA PCR products confirmed that ten of of the isolates were B. gladioli and the remaining 15 were an unidentified Burkholderia species. Sequencing the gene encoding for b-subunit polypeptide of DNA gyrase (gyrB) did not assist identification of these isolates. This study suggests that other Burkholderia species are involved with bacterial softrot of saffron in Sardinia, and further studies are in progress to verify this hypothesis

Identification and characterization of Burkholderia isolates obtained from bacterial rot of saffron (<I>Crocus sativus</I> L.) grown in Italy

<p>Twenty five isolates of <em>Burkholderia gladioli</em>, the causal agent of a bacterial disease recently reported on saffron (<em>Crocus sativus </em>L.) grown in central Sardinia (Italy), were characterized using different approaches. The characteristic symptoms of the disease on saffron plants were rot of emerging shoots and leaves and spots on leaves and corms. In the field, the disease was destructive and reduced flowering by about 80%. Two types of colonies of bacteria cultured from affected plants were selected on the basis of their characteristic morphology and pigment production on nutrient-glucose-agar. One type was round, wrinkled, and producing yellowish pigment; while the second was round, smooth and without pigment. All 25 selected isolates were pathogenic on saffron leaves and corms. Ten were pathogenic on gladiolus and lily leaves. None of the tested isolates was pathogenic on onion plants. The isolates were characterized by conventional tests, Biolog, PCR and PCR-RFLP analysis. Conventional tests and PCR identified all isolates as <em>B. gladioli</em>. PCR-RFLP analysis of 16S rDNA products digested with the three restriction enzymes <em>Alu </em>I, <em>Dde </em>I and <em>Bss </em>KI, identified ten of the isolates as <em>B. gladioli </em>pv. <em>gladioli</em>. Sequencing and comparison of the 16S rDNA PCR products confirmed that ten of of the isolates were <em>B. gladioli </em>and the remaining 15 were an unidentified <em>Burkholderia </em>species. Sequencing the gene encoding for b-subunit polypeptide of DNA gyrase (<em>gyrB</em>) did not assist identification of these isolates. This study suggests that other <em>Burkholderia </em>species are involved with bacterial softrot of saffron in Sardinia, and further studies are in progress to verify this hypothesis.</p

Studies on the susceptibility of pruning wounds to infection by fungi involved in grapevine wood diseases in Italy

The susceptibility of grapevine annual pruning wounds to Phaeomoniella chlamydospora, Phaeoacremonium aleophilum and Diplodia seriata was investigated over three years (2005–2007) in a 15 year-old vineyard, cv. Sauvignon blanc. Vines were pruned each year in January, February and March and the wounds were inoculated weekly with conidial suspensions, and with sterile water as a control. Penetration of the fungi into the wood was assessed after 4 weeks by plating pieces of host tissue on agar medium. The susceptibility of annual pruning wounds, expressed as the infection percentages of inoculated spurs, varied with both the trial year and the fungus inoculated. Average infection percentages of inoculated spurs in the three years were respectively 14.7, 38.5 and 50.9% for Pa. chlamydospora, 31.7, 32.2 and 49.4% for Pm. aleophilum and 84.2, 43.8 and 40.9% for D. seriata. The period of pruning was significant for the infection percentages of all fungi in 2005, and for D. seriata in 2006. Natural infection of control spurs by Pa. chlamydospora (2, 4.4, and 11.7% of spurs in the three years respectively) and by Pm. aleophilum (0.3, 1.8, and 6.4%) began when average weekly temperatures stabilized around 10°C, while infection by D. seriata (12.2, 12 and 18.3% in the same period) occurred even below that threshold. Higher infection percentages of both artificially and naturally infected spurs in 2007 were probably due to the higher temperatures recorded in February and March (besides the use of a more efficient selective medium for the isolation of Pa. chlamydospora and Pm. aleophilum). Only artificial infections with D. seriata showed an opposite trend that cannot be explained by the weather data. Infection of one-year-old wood appeared to be an important factor in disease spread. Spurs remained liable to infections with any of the fungi for up to 4 months after pruning, and isolation percentages could be fairly high also in late spring. As a consequence, the planning of pruning does not seem to be an effective means to counteract the wood diseases caused by these fungi

Searching for an American foulbrood early detection threshold by the determination of <i>Paenibacillus larvae</i> spore load in worker honey bees

In the present study, the relationship between Paenibacillus larvae (White) spore load per adult bee and the appearance of American Foulbrood (AFB) symptoms in the colony was investigated. A total of 44 beehives from 8 apiaries located in Sardinia island (Italy) were involved in this research. Adult bee samples were collected from colonies either showing or lacking the disease symptoms, and the average number of P. larvae spores per adult bee was determined through microbiological culture, followed by bacterial identification through a combination of morphological, biochemical and genetic assays. Correspondence between the detection of disease symptoms in the hives and the number of spores per adult bee samples was obtained. In line with previous studies, the outcome of the present investigation supports the employment of adult bee spore load determination as a tool to get information on the actual health status of the colony. This allows an early detection of AFB disease, especially when clinical symptoms in colonies are still not observable. In addition, the results suggest that a level around 3,000 P. larvae spores per adult bee, obtained with our experimental conditions, might represent a threshold for the appearance of clinical symptoms in the colony