Genetic Transformation of Passionflower and Evaluation of R(1) and R(2) Generations for Resistance to Cowpea aphid borne mosaic virus

We report on the production and evaluation of passionflower transgenic lines for resistance to Cowpea aphid borne mosaic virus (CABMV). Genetic transformation was done using Agrobacterium tumefaciens and transgene integration was confirmed by Southern blot analyses, resulting in nine transgenic lines for `IAC 275` and three for `IAC 277`. Transgenic lines were clonally propagated and evaluated for resistance to CABMV After the third inoculation, under higher inoculum pressure, only propagated plants of the transgenic line T16 remained asymptomatic, indicating a high resistance to infection with CABMV. This transgenic line was self-pollinated and the RI generation was evaluated together with the RI generation of another resistant transgenic line (T2) identified previously. Plants were inoculated with CABMV by means of viruliferous Myzus nicotianae. All 524 T2R(1) plants became infected, whereas 13 of 279 T16R(1) remained asymptomatic after four successive inoculations. A TI6R(2) generation was obtained and plants were inoculated with CABMV mechanically or by aphids. After successive inoculations, 118 of 258 plants were symptomless, suggesting that the resistance to CABMV was maintained in the plant genome as the homozygous condition was achieved. Five selected resistant TI6R(2) plants which contained the capsid protein gene are being crossed for further analyses.

Chitinolytic activity of endophytic Streptomyces and potential for biocontrol

Biological sources for the control of plant pathogenic fungi remain an important objective for sustainable agricultural practices. Actinomycetes are used extensively in the pharmaceutical industry and agriculture owing to their great diversity in enzyme production. In the present study, therefore, we evaluated chitinase production by endophytic actinomycetes and the potential of this for control of phytopathogenic fungi. Endophytic Streptomyces were grown on minimum medium supplemented with chitin, and chitinase production was quantified. The strains were screened for any activity towards phytopathogenic fungi and oomycetes by a dual-culture in vitro assay. The correlation between chitinase production and pathogen inhibition was calculated and further confirmed on Colletotrichum sublineolum cell walls by scanning electron microscopy. This paper reports a genetic correlation between chitinase production and the biocontrol potential of endophytic actinomycetes in an antagonistic interaction with different phytopathogens, suggesting that this control could occur inside the host plant. A genetic correlation between chitinase production and pathogen inhibition was demonstrated. Our results provide an enhanced understanding of endophytic Streptomyces and its potential as a biocontrol agent. The implications and applications of these data for biocontrol are discussed.

Adaptation of an apple sooty blotch and flyspeck warning system for the Upper Midwest United States

A warning system for sooty blotch and flyspeck (SBFS) of apple, developed in the southeastern United States, uses cumulative hours of leaf wetness duration (LWD) to predict the timing of the first appearance of signs. In the Upper Midwest United States, however, this warning system has resulted in sporadic disease control failures. The purpose of the present study was to determine whether the warning system`s algorithm could be modified to provide more reliable assessment of SBFS risk. Hourly LWD, rainfall, relative humidity (RH), and temperature data were collected from orchards in Iowa, North Carolina, and Wisconsin in 2005 and 2006. Timing of the first appearance of SBFS signs was determined by weekly scouting. Preliminary analysis using scatterplots and boxplots suggested that Cumulative hours of RH >= 97% could be a useful predictor of SBFS appearance. Receiver operating characteristic curve analysis was used to compare the predictive performance of cumulative LWD and cumulative hours of RH >= 97%. Cumulative hours of RH >= 97% was a more conservative and accurate predictor than cumulative LWD for 15 site years in the Upper Midwest, but not for four site years in North Carolina. Performance of the SBFS warning system in the Upper Midwest and climatically similar regions may be improved if cumulative hours of RH >= 97% were substituted for cumulative LWD to predict the first appearance of SBFS.

Overwintering of Sclerotium rolfsii and S-rolfsii var. delphinii in different latitudes of the United States

Previously known only from the southern United States, hosta petiole rot recently appeared in the northern United States. Sclerotium rolfsii var. delphinii is believed to be the predominant petiole rot pathogen in the northern United States, whereas S. rolfsii is most prevalent in the southern United States. In order to test the hypothesis that different tolerance to climate extremes affects the geographic distribution of these fungi, the survival of S. rolfsii and S. rolfsii var. delphinii in the northern and southeastern United States was investigated. At each of four locations, nylon screen bags containing sclerotia were placed on the surface of bare soil and at 20-cm depth. Sclerotia were recovered six times from November 2005 to July 2006 in North Dakota and Iowa, and from December 2005 to August 2006 in North Carolina and Georgia. Survival was estimated by quantifying percentage of sclerotium survival on carrot agar. Sclerotia of S. rolfsii var. delphinii survived until at least late July in all four states. In contrast, no S. rolfsii sclerotia survived until June in North Dakota or Iowa, whereas 18.5% survived until August in North Carolina and 10.3% survived in Georgia. The results suggest that inability to tolerate low temperature extremes limits the northern range of S. rolfsii.

Spatial heterogeneity of leaf wetness duration in apple trees and its influence on performance of a warning system for sooty blotch and flyspeck

To determine the effect of sensor placement on the performance of a disease-warning system for sooty blotch and flyspeck (SBFS), we measured leaf wetness duration (LWD) at 12 canopy positions in apple trees, then simulated operation of the disease-warning system using LWD measurements from different parts of the canopy. LWD sensors were placed in four trees within one Iowa orchard during two growing seasons, and in one tree in each of four orchards during a single growing season. The LWD measurements revealed substantial heterogeneity among sensor locations. In all data sets, the upper, eastern portion of the canopy had the longest mean daily LWD, and was the first site to form dew and the last to dry. The lower, western portion of the canopy averaged about 3 It less LWD per day than the top of the canopy, and was the last zone where dew formed and the first to dry off. On about 25% of nights when dew occurred in the top of the canopy, no dew formed in the lower, western canopy. Intracanopy variability of LWD was more pronounced when dew was the sole source of wetness than on days when rainfall occurred. Daily LWD in the upper, eastern portion of the canopy was slightly less than reference measurements made at a 0.7-m height over turfgrass located near the orchard. When LWD measurements from several canopy positions were input to the SBFS warning system, timing of occurrence of a fungicide-spray threshold varied by as much as 30 days among canopy positions. Under Iowa conditions, placement of an LWD sensor at an unobstructed site over turfgrass was a fairly accurate surrogate for the wettest part of the canopy. Therefore, such an extra-canopy LWD sensor might be substituted for a within-canopy sensor to enhance operational reliability of the SBFS warning system.

Citrus Sudden Death Is Transmitted by Graft-Inoculation and Natural Transmission Is Prevented by Individual Insect-Proof Cages

Citrus sudden death (CSD) transmission was studied by graft-inoculation and under natural conditions. Young sweet orange trees on Rangpur rootstock were used as indicator plants. They were examined regularly for one or two characteristic markers of CSD: (i) presence of a yellow-stained layer of thickened bark on the Rangpur rootstock, and (ii) infection with the CSD-associated marafivirus. Based on these two markers, transmission of CSD was obtained, not only when budwood for graft-inoculation was taken from symptomatic, sweet orange trees on Rangpur, but also when the budwood sources were asymptomatic sweet orange trees on Cleopatra mandarin, indicating that the latter trees are symptomless carriers of the CSD agent. For natural transmission, 80 young indicator plants were planted within a citrus plot severely affected by CSD. Individual insect-proof cages were built around 40 indicator plants, and the other 40 indicator plants remained uncaged. Only two of the 40 caged indicator plants were affected by CSD, whereas 17 uncaged indicator plants showed CSD symptoms and were infected with the marafivirus. An additional 12 uncaged indicator plants became severely affected with citrus variegated chlorosis and were removed. These results strongly suggest that under natural conditions, CSD is transmitted by an aerial vector, such as an insect, and that the cages protected the trees against infection by the vector.

Soil solarization for weed control in carrot

Soil solarization is a technique used for weed and plant disease control in regions with high levels of solar radiation. The effect of solarization (0, 3, 6, and 9 weeks) upon weed populations, carrot (Daucus carota L. cv. Brasília) yield and nematode infestation in carrot roots was studied in São Luís (2º35' S; 44º10' W), MA, Brazil, using transparent polyethylene films (100 and 150 mm of thickness). The maximum temperature at 5 cm of depth was about 10ºC warmer in solarized soil than in control plots. In the study 20 weed types were recorded. Solarization reduced weed biomass and density in about 50% of weed species, including Cyperus spp., Chamaecrista nictans var. paraguariensis (Chod & Hassl.) Irwin & Barneby, Marsypianthes chamaedrys (Vahl) O. Kuntze, Mitracarpus sp., Mollugo verticillata L., Sebastiania corniculata M. Arg., and Spigelia anthelmia L. Approximately 40% of species in the weed flora were not affected by soil mulching. Furthermore, seed germination of Commelina benghalensis L. was increased by soil solarization. Marketable yield of carrots was greater in solarized soil than in the unsolarized one. It was concluded that solarization for nine weeks increases carrot yield and is effective for controlling more than half of the weed species recorded. Mulching was not effective for controlling root-knot nematodes in carrot.

Current knowledge on the Ralstonia solanacearum type III secretion system

R. solanacearum was ranked in a recent survey the second most important bacterial plant pathogen, following the widely used research model Pseudomonas syringae (Mansfield et al., 2012). The main reason is that bacterial wilt caused by R. solanacearum is the world"s most devastating bacterial plant disease (http://faostat.fao.org), threatening food safety in tropical and subtropical agriculture, especially in China, Bangladesh, Bolivia and Uganda (Martin and French, 1985). This is due to the unusually wide host range of the bacterium, its high persistence and because resistant crop varieties are unavailable. In addition, R. solanacearum has been established as a model bacterium for plant pathology thanks to pioneering molecular and genomic studies (Boucher et al., 1985; Cunnac et al., 2004b; Mukaihara et al., 2010; Occhialini et al., 2005; Salanoubat et al., 2002). As for many bacterial pathogens, the main virulence determinant in R. solanacearum is the type III secretion system (T3SS) (Boucher et al., 1994), which injects a number of effector proteins into plant cells causing disease in hosts or an hypersensitive response in resistant plants. In this article we discuss the current state in the study of the R. solanacearum T3SS, stressing the latest findings and future perspectives.

Antibacterial activity of essential oils on Xanthomonas vesicatoria and control of bacterial spot in tomato

The objective of this work was to evaluate the effects of plant essential oils (EOs) on the growth of Xanthomonas vesicatoria, on bacterial morphology and ultrastructure, and on the severity of tomato bacterial spot. EOs from citronella, clove, cinnamon, lemongrass, eucalyptus, thyme, and tea tree were evaluated in vitro at concentrations of 0.1, 1.0, 10, and 100% in 1.0% powdered milk. The effect of EOs, at 0.1%, on the severity of tomato bacterial spot was evaluated in tomato seedlings under greenhouse conditions. The effects of citronella, lemongrass, clove, and tea tree EOs, at 0.1%, on X. vesicatoria cells were evaluated by transmission electron microscopy. All EOs showed direct toxic effect on the bacteria at a 10%-concentration in vitro. Under greenhouse conditions, the EOs of clove, citronella, tea tree, and lemongrass reduced disease severity. EOs of clove and tea tree, and streptomycin sulfate promoted loss of electron-dense material and alterations in the cytoplasm, whereas EO of tea tree promoted cytoplasm vacuolation, and those of citronella, lemongrass, clove, and tea tree caused damage to the bacterial cell wall. The EOs at a concentration of 0.1% reduce the severity of the disease.

A risk infection simulation model for fusarium head blight of wheat

Fusarium Head Blight (FHB) is a disease of great concern in wheat (Triticum aestivum). Due to its relatively narrow susceptible phase and environmental dependence, the pathosystem is suitable for modeling. In the present work, a mechanistic model for estimating an infection index of FHB was developed. The model is process-based driven by rates, rules and coefficients for estimating the dynamics of flowering, airborne inoculum density and infection frequency. The latter is a function of temperature during an infection event (IE), which is defined based on a combination of daily records of precipitation and mean relative humidity. The daily infection index is the product of the daily proportion of susceptible tissue available, infection frequency and spore cloud density. The model was evaluated with an independent dataset of epidemics recorded in experimental plots (five years and three planting dates) at Passo Fundo, Brazil. Four models that use different factors were tested, and results showed all were able to explain variation for disease incidence and severity. A model that uses a correction factor for extending host susceptibility and daily spore cloud density to account for post-flowering infections was the most accurate explaining 93% of the variation in disease severity and 69% of disease incidence according to regression analysis.

Weather based warning systems for bean angular-leaf-spot and anthracnose

Data available in the literature were used to develop a warning system for bean angular leaf spot and anthracnose, caused by Phaeoisariopsis griseola and Colletotrichum lindemuthianum, respectively. The model is based on favorable environmental conditions for the infectious process such as continuous leaf wetness duration and mean air temperature during this subphase of the pathogen-host relationship cycle. Equations published by DALLA PRIA (1977) showing the interactions of those two factors on the disease severity were used. Excell spreadsheet was used to calculate the leaf wetness period needed to cause different infection probabilities at different temperature ranges. These data were employed to elaborate critical period tables used to program a computerized electronic device that records leaf wetness duration and mean temperature and automatically shows the daily disease severity value (DDSV) for each disease. The model should be validated in field experiments under natural infection for which the daily disease severity sum (DDSS) should be identified as a criterion to indicate the beginning and the interval of fungicide applications to control both diseases.

Application and validation of a warning system for grapevine downy mildew controlusing fungicides

Most warning systems for plant disease control are based on Vinho, in Bento Gonçalves - RS, during the growing seasons 2000/ weather models dependent on the relationships between leaf wetness 01, 2002/03 and 2003/2004, using the grape cultivar Isabel. The duration and mean air temperature in this period considering the conventional system used by local growers was compared with the target disease intensity. For the development of a warning system to new warning system by using different cumulative daily disease severity control grapevine downy mildew, the equation generated by Lalancette values (CDDSV) as the criterion to schedule fungicide application and et al. (7) was used. This equation was employed to elaborate a critical reapplication. In experiments conducted in 2003/04, CDDSV of 12 - period table and program a computerized device, which records, though 14 showed promising to schedule the first spraying and the interval electronic sensors, leaf wetness duration, mean temperature in this between fungicide applications, reducing by 37.5% the number of period and automatically calculates the daily value of probability of applications and maintaining the same control efficiency in leaves infection occurrence. The system was validated at Embrapa Uva e and bunches, similarly to the conventional system.

Influence of temperature and leaf wetness duration in the severity of Cercospora leaf spot of beet

ABSTRACT In the present study, the influence of temperature (15, 20, 25, 30 and 35°C) and leaf wetness period (6, 12, 24 and 48 hours) on the severity of Cercospora leaf spot of beet, caused by Cercospora beticola, was studied under controlled conditions. Lesion density was influenced by temperature and leaf wetness duration (P<0.05). Data were subjected to nonlinear regression analysis. The generalized beta function was used for fitting the disease severity and temperature data, while a logistic function was chosen to represent the effect of leaf wetness on the severity of Cercospora leaf spot. The response surface resultant of the product of the two functions was expressed as ES = 0.0001105 * (((x-8)2.294387) * ((36-x)0.955017)) * (0.39219/(1+25.93072 * exp (-0.16704*y))), where: ES represents the estimated severity value (0.1); x, the temperature (ºC) and y, the leaf wetness duration (hours). This model should be validated under field conditions to assess its use as a computational forecast system for Cercospora leaf spot of beet.

Evidence that nematodes may vector the soft rot-causing enterobacterial phytopathogens

Bacterial soft rot is a globally significant plant disease that causes major losses in the production of many popular crops, such as potato. Little is known about the dispersal and ecology of soft-rot enterobacteria, and few animals have been identified as vectors for these pathogens. This study investigates whether soil-living and bacterial-feeding nematodes could act as vectors for the dispersal of soft-rot enterobacteria to plants. Soft-rot enterobacteria associated with nematodes were quantified and visualized through bacterial enumeration, GFP-tagging, and confocal and electron scanning microscopy. Soft-rot enterobacteria were able to withstand nematode grazing, colonize the gut of Caenorhabditis elegans and subsequently disperse to plant material while remaining virulent. Two nematode species were also isolated from a rotten potato sample obtained from a potato storage facility in Finland. Furthermore, one of these isolates (Pristionchus sp. FIN-1) was shown to be able to disperse soft-rot enterobacteria to plant material. The interaction of nematodes and soft-rot enterobacteria seems to be more mutualistic rather than pathogenic, but more research is needed to explain how soft-rot enterobacteria remain viable inside nematodes.

Caracterização da mutagênese por inserção do retrotransposon Tos17 em genótipos de arroz

O aumento do potencial produtivo da cultura de arroz via melhoramento genético está principalmente relacionado ao rendimento, a qualidade de grãos e a obtenção de plantas resistentes a doenças e pragas. Neste caso, deve ser explorada a variabilidade genética natural ou induzida através de agentes mutagênicos físicos, químicos ou biológicos. A vantagem do uso de mutagênicos biológicos, como os transposons e os retrotransposons, é que ao serem inseridos podem interrompem um gene causando uma mutação. Esta retrotransposição deixa uma marca que possibilita a identificação molecular do local de inserção. No presente trabalho, foi utilizada a estratégia de mutagênese insercional através de eventos de transposição do retrotransposon Tos17, induzido por cultura in vitro. A análise de diferentes genótipos de arroz é muito importante para avaliar se a transposição ocorre de forma similar em genótipos distintos. Foram avaliados calos embriogênicos de cultivares que têm um histórico de variabilidade genética em homozigose, linhagens obtidas através de cruzamentos com espécies silvestres e ecótipos de arroz vermelho, submetidos a 6 meses de cultura in vitro. O método escolhido para avaliar o número de cópias de Tos17 em calos embriogênicos foi a quantificação relativa por PCR em tempo real. A identificação dos genes mutados pela inserção do retrotransposon foi feita através do isolamento e amplificação das seqüências que flanqueiam os insertos de Tos17. O resultado deste experimento indica um aumento no número de cópias de Tos17 em 8 dos 21 genótipos avaliados. O seqüenciamento dos fragmentos de DNA que flanqueiam os insertos do retrotransposon Tos17, indicaram alta similaridade com seqüências genômicas não codificantes de arroz.