Effect of Hot Water Treatments on Eradication of «Phaeomoniella chlamydospora» and «Phaeoacremonium inflatipes» from Dmant Grapevine Wood

Hot water treatments were applied to eradicate Phaeoacremonium inflatipes and Phaeomoniella chlamydospora from dormant grapevine wood. A thirty-minute hot water treatment at 51°C did not eliminate these pathogens from dormant wood cuttings. Cuttings first inoculated with Pa. chlamydospora or Pm. inflatipes or both fungi, and then subjected to a hot water treatment were either incubated in crispers, or planted for six to eight weeks. Vascular discoloration was scored followed by isolation from the cuttings onto potato dextrose agar amended with 0.1 g l-1 tetracycline (PDA-tet). Isolations confirmed the presence of the pathogens in the inoculated, hot-water treated cuttings as well as in the inoculated, untreated control cuttings. This finding, along with earlier research on the direct effect of hot water on the mycelium of these species, leads to the conclusion that hot water treatments are ineffective in eliminating vine decline pathogens from dormant wood

Association of Spores of «Phaeomoniella chlamydospora», «Phaeoacremonium inflatipes», and «Pm. aleophilum» with Grapevine Cordons in California

Esca (black measles) of grapevine has long been known to occur wherever grapes are grown. Phaeomoniella chlamydospora and two species of Phaeoacremonium, Pm. inflatipes and Pm. aleophilum, have been associated with esca and Petri grapevine decline in major production regions of California. Though present in symptomatic grapevines and capable of causing foliar symptoms of esca, Phaeomoniella chlamydospora does not cause the typical symptoms on fruit. However, trapping studies showed that spores of Pa. chlamydospora, Pm. inflatipes and Pm. aleophilum were captured throughout the year in vineyards ranging from the north California coast to the southern San Joaquin Valley. They can be considered airborne fungi capable of being water-splashed by pruning or other wounds during part of their biological cycle. Trapping of spores coincided with rainfall events for Pa. chlamydospora and Pm. inflatipes, and to a lesser degree for Pm. aleophilum. However, this last species was trapped during periods of time when rainfall did not occur and was trapped longer into the summer

Histological Investigations on Infection of Grape Roots and Shoots by «Phaeoacremonium» spp.

Tissue-cultured grapevines cv. Cabernet Sauvignon were used to determine whether injury is a prerequisite to penetration of shoot and root tissue by Phaeoacremonium spp. and to ascertain the path of invasion inside the host. Uninjured and injured roots and shoots were inoculated with P. inflatipes and P. aleophilum at approximately 106 spores ml-1. Isolation and scanning electron microscopy observations showed that Phaeoacremonium spp. was capable of penetrating uninjured roots and shoots. Light microscopy observations of paraffin embedded sections showed that rapid spread of the fungus in roots was through the vascular tissues and intercellular spaces of the cortex. In the shoots, spread of the fungus was initially through the intercellular spaces of the epidermis, cortex, and pith; rapid spread apparently occurred in the intercellular spaces of the pith. Spores were seen in the pith area as well as in the xylem. Remote from the point of inoculation, hyphae were seen in the epidermis, cortex, pith, and vascular tissues without noticeable change in appearance of the invaded cells. Symptoms of the disease were observed in some plants 2 months after inoculation but isolations made in symptomless plants after 2 weeks showed the presence of the fungus in all parts of the plant

Adaptive genomic structural variation in the grape powdery mildew pathogen, Erysiphe necator.

BackgroundPowdery mildew, caused by the obligate biotrophic fungus Erysiphe necator, is an economically important disease of grapevines worldwide. Large quantities of fungicides are used for its control, accelerating the incidence of fungicide-resistance. Copy number variations (CNVs) are unbalanced changes in the structure of the genome that have been associated with complex traits. In addition to providing the first description of the large and highly repetitive genome of E. necator, this study describes the impact of genomic structural variation on fungicide resistance in Erysiphe necator.ResultsA shotgun approach was applied to sequence and assemble the genome of five E. necator isolates, and RNA-seq and comparative genomics were used to predict and annotate protein-coding genes. Our results show that the E. necator genome is exceptionally large and repetitive and suggest that transposable elements are responsible for genome expansion. Frequent structural variations were found between isolates and included copy number variation in EnCYP51, the target of the commonly used sterol demethylase inhibitor (DMI) fungicides. A panel of 89 additional E. necator isolates collected from diverse vineyard sites was screened for copy number variation in the EnCYP51 gene and for presence/absence of a point mutation (Y136F) known to result in higher fungicide tolerance. We show that an increase in EnCYP51 copy number is significantly more likely to be detected in isolates collected from fungicide-treated vineyards. Increased EnCYP51 copy numbers were detected with the Y136F allele, suggesting that an increase in copy number becomes advantageous only after the fungicide-tolerant allele is acquired. We also show that EnCYP51 copy number influences expression in a gene-dose dependent manner and correlates with fungal growth in the presence of a DMI fungicide.ConclusionsTaken together our results show that CNV can be adaptive in the development of resistance to fungicides by providing increasing quantitative protection in a gene-dosage dependent manner. The results of this work not only demonstrate the effectiveness of using genomics to dissect complex traits in organisms with very limited molecular information, but also may have broader implications for understanding genomic dynamics in response to strong selective pressure in other pathogens with similar genome architectures

Recovery of «Phaemoniella chlamydospora» and «Phaeoacremonium inflatipes» from Soil and Grapevine Tissues

Rose Bengal Chloramphenicol Agar (RBCA) appears to be a suitable media for isolating Phaeoacremonium spp. and Phaeomoniella chlamydospora from soil, spore traps and plant tissue. Using the soil-plate method, populations of these organisms were recovered from the soil and surfaces of plant tissue from many different regions of California. In addition, in a few vineyard sites these fungi were recovered from dried plant sap, which had oozed from grapevine girdling wounds and from standing water under grapevine drip systems. RBCA, along with a filtering system, is a useful tool in determining the presence of Petri disease pathogens in vineyard soils, water, and plant tissues. This research presents the first report of the recovery of Phaeoacremonium inflatipes from soil and standing water under grapevines

Rootstock Susceptibility to «Phaeomoniella chlamydospora» and «Phaeoacremonium» spp.

Twenty healthy cuttings each were inoculated artificially with Phaeomoniella chlamydospora, Phaeoacremonium inflatipes and Phaeoacremonium aleophilum. After the formation of callus, they were planted in pots. Approximately one year after inoculation, disease occurrence was recorded as the length of brown vascular streaking from the base of plant toward the shoot tip. Discolored areas were cultured on potato dextrose agar amended with tetracycline hydrochloride (PDA-tet) and pathogens were re-isolated. Inoculation with Pa. chlamydospora showed that rootstocks 3309, 420A, 110R, 5C, Schwarzmann, St. George, and Salt Creek were least susceptible, while 99R, 39-16, Freedom, Riparia Gloire, 140Ru, 16-16 and 1103 were most susceptible. When inoculated with Pm. inflatipes, 16-16, 3309, AXR1, Salt Creek, 110R, 5C, Freedom and 140Ru were least susceptible, while 420A, St. George, 161-49, and Harmony were most susceptible. Inoculation with Pm. aleophilum showed that 1103, 420A, Harmony, and Salt Creek were least susceptible, while 110R, SO4, 39-16 and 161-49 were most susceptible. The susceptibility of the rootstocks and the occurrence of vine decline in the field in California did not appear to be well correlated because 3309, 101-14, 5C, and 110R were always most susceptible. But these rootstocks are the most widely planted, and hence the natural occurrence of the disease is probably skewed towards these rootstocks. However, since no resistance was detected in our study, even though there was a wide range of susceptibility, the degree of rootstock susceptibility may not be such an important factor in disease expression under natural conditions

Effects of punctuated heat stress on the grapevine powdery mildew pathogen, Erysiphe necator

Grapevine powdery mildew, caused by the ascomycete Erysiphe necator, is a major threat to grapes worldwide. Despite its global impact on grape production, E. necator is sensitive to adverse environmental conditions, such as excess heat, free water and UV radiation. Using detached leaf co-culture assays, 3-d-old single colonies of E. necator were exposed to 1, 2 or 3 consecutive d of punctuated heat stress. While there was a consistent decrease in colony growth after a single heating event, there were little to no significant effects from subsequent heating events. Similar effects were observed on the latent period of the pathogen, with a large initial effect from the first heat treatment and small marginal effects from subsequent heat treatments. Erysiphe necator colonies growing on live pot-grown plants were affected similarly by consecutive heat stress events. These data suggest that E. necator is more adaptable to environmental stress than previously recognized

Epidemiology of Dengue Virus in Iquitos, Peru 1999 to 2005: Interepidemic and Epidemic Patterns of Transmission

To develop prevention (including vaccines) and control programs for dengue fever, a significant mosquito-borne disease in the tropics, there is an urgent need for comprehensive long term field epidemiological studies. We report results from a study that monitored ∼2,400 school children and some adult family members for dengue infection at 6 month intervals from 1999 to 2005, in the Amazonian city of Iquitos, Peru. At enrollment, ∼80% of the participants had a previous infection with DENV serotypes 1 and 2 or both. During the first 15 months, about 3 new infections for every 100 participants were observed among the study participants. In 2001, DENV-3, a serotype not previously observed in the region, invaded Iquitos in a process characterized by 3 distinct periods: amplification over at least a 5–6 month period, replacement of previously circulating serotypes, and epidemic transmission when incidence peaked. Incidence patterns of new infections were geographically distinct from baseline prevalence rates prior to arrival of DENV-3, but closely mirrored them during the invasion. DENV transmission varied geographically corresponding to elevated mosquito densities. The invasion of a novel serotype is often characterized by 5–6 months of silent transmission before traditional surveillance programs detect the virus. This article sets the stage for subsequent publications on dengue epidemiology

Spatial Dimensions of Dengue Virus Transmission across Interepidemic and Epidemic Periods in Iquitos, Peru (1999–2003)

To target prevention and control strategies for dengue fever, it is essential to understand how the virus travels through the city. We report spatial analyses of dengue infections from a study monitoring school children and adult family members for dengue infection at six-month intervals from 1999–2003, in the Amazonian city of Iquitos, Peru. At the beginning of the study, only DENV serotypes 1 and 2 were circulating. Clusters of infections of these two viruses were concentrated in the northern region of the city, where mosquito indices and previous DENV infection were both high. In 2002, DENV-3 invaded the city, replacing DENV-1 and -2 as the dominant strain. During the invasion process, the virus spread rapidly across the city, at low levels. After this initial phase, clusters of infection appeared first in the northern region of the city, where clusters of DENV-1 and DENV-2 had occurred in prior years. Most of the clusters we identified had radii >100 meters, indicating that targeted or reactive treatment of these high-risk areas might be an effective proactive intervention strategy. Our results also help explain why vector control within 100 m of a dengue case is often not successful for large-scale disease prevention

Landscape Ecology of Sylvatic Chikungunya Virus and Mosquito Vectors in Southeastern Senegal

The risk of human infection with sylvatic chikungunya (CHIKV) virus was assessed in a focus of sylvatic arbovirus circulation in Senegal by investigating distribution and abundance of anthropophilic Aedes mosquitoes, as well as the abundance and distribution of CHIKV in these mosquitoes. A 1650 km2 area was classified into five land cover classes: forest, barren, savanna, agriculture and village. A total of 39,799 mosquitoes was sampled from all classes using human landing collections between June 2009 and January 2010. Mosquito diversity was extremely high, and overall vector abundance peaked at the start of the rainy season. CHIKV was detected in 42 mosquito pools. Our data suggest that Aedes furcifer, which occurred abundantly in all land cover classes and landed frequently on humans in villages outside of houses, is probably the major bridge vector responsible for the spillover of sylvatic CHIKV to humans