Megan M. Dewdney

Spatial Distribution of Venturia inaequalis Airborne Ascospores in Orchards.

ABSTRACT Apple scab (Venturia inaequalis) causes important economic losses in many apple production areas of the world. The disease is controlled by numerous fungicide applications regardless of the presence of ascospores in the orchard. Airborne ascospore concentration (AAC) can be measured in real time to time fungicide applications. However, the level of heterogeneity of the AAC in commercial orchards was unknown. Consequently, the spatial distribution of V. inaequalis ascospores was studied in a commercial apple orchard of 0.43 ha. The potential ascospore dose (PAD) and AAC were measured in 40 quadrats each of 108 m(2). In each quadrat, the AAC was monitored during the major rain events in spring 1999 and 2000 using spore samplers. The variance-to-mean ratio for the PAD and for most of the AAC sampling dates was >1, indicating an aggregated pattern of distribution. None of the frequency distributions of the most important ascospore ejection events followed the Poisson probability distribution, indicating that the pattern of distribution was not random. For all events, AAC had an aggregated pattern of distribution as suggested by the negative binomial distribution. The PAD followed neither the Poisson nor the negative binomial distribution. Geostatistical analyses confirmed the aggregated pattern of distribution. The cultivars had an effect on the PAD and AAC distribution pattern, but both PAD and AAC were not uniformly distributed within a block of the same cultivar. Therefore, the number, location, and height of samplers required to estimate AAC in orchards need to be investigated before using information on AAC for decision making.

A statistical comparison of the blossom blight forecasts of MARYBLYT and Cougarblight with receiver operating characteristic curve analysis

ABSTRACT Blossom blight forecasting is an important aspect of fire blight, caused by Erwinia amylovora, management for both apple and pear. A comparison of the forecast accuracy of two common fire blight forecasters, MARYBLYT and Cougarblight, was performed with receiver operating characteristic (ROC) curve analysis and 243 data sets. The rain threshold of Cougarblight was analyzed as a separate model termed Cougarblight and rain. Data were used as a whole and then grouped into geographic regions and cultivar susceptibilities. Frequency distributions of cases and controls, orchards or regions (depending on the data set), with and without observed disease, respectively, in all data sets overlapped. MARYBLYT, Cougarblight, and Cougarblight and rain all predicted blossom blight infection better than chance (P = 0.05). It was found that the blossom blight forecasters performed equivalently in the geographic regions of the east and west coasts of North America and moderately susceptible cultivars based on the 95% confidence intervals and pairwise contrasts of the area under the ROC curve. Significant differences (P < 0.05) between the forecasts of Cougarblight and MARYBLYT were found with pairwise contrasts in the England and very susceptible cultivar data sets. Youdens index was used to determine the optimal cutpoint of both forecasters. The greatest sensitivity and specificity for MARYBLYT coincided with the use of the highest risk threshold for predictions of infection; with Cougarblight, there was no clear single risk threshold across all data sets.

Diaporthe species occurring on citrus in China

Diaporthe species are important pathogens of a wide range of hosts including many economically important crops. Diaporthe citri is generally accepted to cause melanose of Citrus fruits, leaves and shoots; stem-end rot of fruits and gummosis of perennial branches. In this study, 147 strains of Diaporthe were isolated from diseased leaves, twigs, fruits and branches of Citrus from Shaanxi, Zhejiang, Guangxi, Fujian, Guangdong, Jiangxi, Hunan, Chongqing and Shanghai and identified by morphology and molecular analysis of nuclear ribosomal internal transcribed spacer (ITS), partial sequences of translation elongation factor 1-α (EF 1-α), tubulin (TUB) and calmodulin (CAL) genes. All strains from Citrus clustered in three clades. Diaporthe citri was the predominant species and is compared with strains from Florida, USA, where this species was first collected and described. Two new taxa differing from D. citri are described as D. citriasiana and D. citrichinensis in this paper. Inoculation tests indicate that all three species are able to cause stem-end rot of fruits, while only D. citri could cause melanose on leaves.

A Rapid Resazurin-Based Microtiter Assay to Evaluate QoI Sensitivity for Alternaria alternata Isolates and Their Molecular Characterization

Chemical management of Alternaria brown spot of citrus is based upon the timely application of site-specific fungicides, many of which are vulnerable to the development of fungicide resistance. A rapid microtiter bioassay based on the colorimetric changes of resazurin (RZ) dye was developed to evaluate the sensitivity of Alternaria alternata to quinone outside inhibitor (QoI) fungicides. Four liquid media (complete medium, minimal medium, potato dextrose broth, and yeast peptone dextrose broth), five conidia concentrations (from 101 to 105 conidia/ ml), and five RZ concentrations (10, 20, 30, 40, and 50 μM) were evaluated. Complete medium at 105 conidia/ml and 40 μM RZ were identified as optimal for measuring RZ reduction. The effective concentration of two QoI fungicides (azoxystrobin and pyraclostrobin) needed to reduce RZ by 50% (EC50) was calculated and compared with those obtained from conidia germination tests on fungicide-amended media. Concordant EC50 values were observed (R2 = 0.923; P < 0.0001) from both methods. Resistant phenotypes were further characterized by the partial sequencing of the cytochrome b gene. Genetic variability associated with the presence or absence of two introns was observed among isolates. The identified resistant isolates had the amino acid substitution G143A, typical of QoI resistance in other fungi.

Baseline Sensitivity of Guignardia citricarpa Isolates from Florida to Azoxystrobin and Pyraclostrobin

Citrus black spot (CBS), caused by Guignardia citricarpa, is an emerging disease in Florida. Fungicide applications are the main control measure worldwide. The in vitro activity and baseline sensitivity of G. citricarpa isolates to quinone outside inhibitor (QoI) fungicides (azoxystrobin and pyraclostrobin) were evaluated. The effective concentration needed to reduce mycelial growth or spore germination by 50% (EC50) was determined for 86 isolates obtained from Florida counties where CBS is found. The effect of salicylhydroxamic acid (SHAM) plus azoxystrobin and pyraclostrobin was also assessed for mycelial growth and conidial germination. The mean EC50 for mycelial growth for azoxystrobin was 0.027 μg/ml and that for pyraclostrobin was significantly lower at 0.007 μg/ml (P < 0.0001). Similarly, the mean EC50 for conidial germination for azoxystrobin was 0.016 μg/ml and that for pyraclostrobin was significantly lower at 0.008 μg/ml (P < 0.0001). There was no effect of SHAM on inhibition of mycelial growth or conidial germination by the QoI fungicides but SHAM slightly affected mycelium inhibition by pyraclostrobin. Cytochrome b was partially sequenced and three group 1 introns were found. One intron was immediately post G143, likely inhibiting resistance-conferring mutations at that site. It is surmised that the QoI resistance risk is low in the Florida G. citricarpa population.

Mating Type and Simple Sequence Repeat Markers Indicate a Clonal Population of Phyllosticta citricarpa in Florida

Phyllosticta citricarpa, the citrus black spot pathogen, was first identified in Florida in March 2010. Subsequently, this pathogen has become established in Florida but can be easily confused with the endemic nonpathogenic citrus endophyte P. capitalensis. In this study, the mating-type (MAT) loci of P. citricarpa and P. capitalensis were identified via draft genome sequencing and were characterized at the structural and sequence levels. P. citricarpa was determined to have an idiomorphic, heterothallic MAT locus structure, whereas P. capitalensis was found to have a single MAT locus consistent with a homothallic mating system. A survey of P. citricarpa isolates from Florida revealed that only the MAT1-2 idiomorph existed in the Floridian population. In contrast, isolates collected from Australia exhibited a 1:1 ratio of MAT1-1 and MAT1-2 isolates. Development and analysis of simple sequence repeat markers revealed a single multilocus genotype (MLG) in the Floridian population (n = 70) and 11 MLG within the Australian population (n = 24). These results indicate that isolates of P. citricarpa from Florida are likely descendent from a single clonal lineage and are reproducing asexually. The disease management focus in Florida will need to be concentrated on the production and dispersal of pycnidiospores.

Distribution of QoI Resistance in Populations of Tangerine-Infecting Alternaria alternata in Florida

Chemical control, based on copper and quinone outside inhibitor (QoI) fungicides, has been essential for the management of brown spot of citrus, caused by Alternaria alternata. However, QoI control failures were detected recently in Florida. From 2008 to 2012, 817 monoconidial isolates of A. alternata from 46 citrus orchards were examined for sensitivity to azoxystrobin (AZ) and pyraclostrobin (PYR). Of the isolates, 57.6% were resistant to both fungicides, with effective concentration to inhibit 50% growth (EC50) values greater than 5 μg/ml for AZ and 1 μg/ml for PYR. The mean EC50 values for sensitive isolates were 0.139 and 0.020 μg/ml for AZ and PYR, respectively. The EC50 values of both fungicides were highly correlated (P < 0.0001), indicating cross resistance. The proportion of resistant isolates differed significantly (P < 0.0001) among cultivars and with QoI application frequency (P < 0.0001). However, resistance was not significantly related (P = 0.364) to disease severity in the field (low, moderate, and high) or isolate virulence (P = 0.397). The molecular basis for QoI resistance was determined for a subset of 235 isolates using polymerase chain reaction restriction fragment length polymorphism of the cytochrome b gene. All resistant isolates showed the point mutation G143A. Based on the presence of one or two introns, isolates were classified as profile I and profile II, respectively. The resistance frequency was significantly higher (P < 0.0001) in isolate profile II, suggesting a higher selection pressure for resistant population profile II.

qPCR Quantification of Pathogenic Guignardia citricarpa and Nonpathogenic G. mangiferae in Citrus

Citrus black spot, a major citrus disease caused by Guignardia citricarpa, was recently introduced in Florida. The nonpathogenic fungal endophyte G. mangiferae is commonly found in the same citrus tissues as G. citricarpa. Quantitative polymerase chain reaction (qPCR) assays based on internal transcribed spacer (ITS)-1 genes were developed to detect, quantify, and distinguish between these morphologically similar organisms in environmental samples. The primer/probe sets GCITS and GMITS were more than 95% efficient in single-set reactions in complex environmental DNA samples. Detection of 10 fg of G. citricarpa and G. mangiferae DNA was possible. Pycnidiospore disruption resulted in detection of single pycnidiospores with 78 (59 to 102; 95% confidence interval [CI]) and 112 (92 to 136; 95% CI) ITS copies for G. citricarpa and G. mangiferae, respectively. Detection was from partially decomposed leaves where fruiting bodies cannot be morphologically distinguished. Temperature and wetting period have significant effects on Guignardia spp. pseudothecia production in leaf litter. Based on relative biomass or the proportion of nuclei detected, G. citricarpa and G. mangiferae respond more strongly to wetting period than temperature. This qPCR assay will provide additional epidemiological data on black spot in tissues where G. citricarpa and G. mangiferae are not easily distinguished.

Transgenic citrus expressing the antimicrobial gene Attacin E (attE) reduces the susceptibility of 'Duncan' grapefruit to the citrus scab caused by Elsinoë fawcettii

Citrus scab, caused by Elsinoë fawcettii (anamorph Sphaceloma fawcettii), is a common foliar fungal disease affecting many citrus cultivars, including grapefruit. No commercial grapefruit cultivar is resistant to scab, and the disease results in severely blemished fruit which reduces its marketability. Transgenic ‘Duncan’ grapefruit trees expressing the antimicrobial attE gene were produced via Agrobacterium-mediated transformation. In in vitro leaf and greenhouse assays, several transgenic-lines had significantly lower susceptibility to E. fawcettii compared to the non-transformed control (P < 0.0001). In the greenhouse studies, sporulation on all transgenic lines except 1 was significantly reduced (P < 0.0001) but the level of sporulation over time did not correspond to disease severity ratings. Lesion size was also significantly reduced on transgenic lines compared to the non-transformed control (P < 0.0001) and the least susceptible line A-23 had the smallest lesions, but in general there was no correlation between lesion size and disease susceptibility. The level of attE mRNA was inversely related to the number of copies detected by Southern blot. The least susceptible line had a single inserted copy of the attE transgene whereas more susceptible lines had multiple copies. Since the attacin mode of action was thought to be specific to Gram-negative bacteria, it was unexpected to find that there was a significant activity against E. fawcettii.

Genetic variation among Phyllosticta strains isolated from citrus in Florida that are pathogenic or nonpathogenic to citrus

Citrus black spot is an emerging disease in Florida since 2010. The causal agent is Phyllosticta citricarpa (teleomorph Guignardia citricarpa), but non-pathogenic P. capitalensis (teleomorph often referred to as G. mangiferae) is often isolated from black spot lesions. Florida isolates of P. citricarpa and P. capitalensis from citrus have not been characterized in detail. In this study, Phyllosticta species isolated from Florida citrus were compared with worldwide isolates using multi-locus sequencing of four conserved loci (rDNA ITS, TEF1, ACT, and GPDH genes). Moreover, the diversity within the two Phyllosticta species was compared based on the same four loci. DNA sequences of P. citricarpa and P. capitalensis were clearly distinct, coinciding with other P. citricarpa and P. capitalensis sequences from different continents. The species showed different population structures in Florida. P. citricarpa isolates did not exhibit genetic variation and were similar to strains from other continents. In contrast, Florida P. capitalensis isolates were distributed over five sequence groups. This study did not point to the potential origin of P. citricarpa and P. capitalensis in Florida. More variable genetic markers and isolates from various continents would be required to track the possible movement of these Phyllosticta species.