Diversity and Clonality in Populations of Phytophthora citrophthora and P. syringae Causing Brown Rot of Citrus in California.

Phytophthora citrophthora and P. syringae are currently the primary causal organisms of brown rot of citrus fruits in California. To possibly find an explanation for the prevalence of the previously minor species P. syringae, we determined the population structures of both pathogens in California using next-generation sequencing and population genomics analyses. Whole-genome sequencing and aligning with newly assembled reference genomes identified 972,266 variants in 132 isolates of P. citrophthora and 422,208 variants in 154 isolates (including 24 from noncitrus tree crops) of P. syringae originating from three major growing regions. The resulting data sets were visualized using principal component analysis, discriminant analysis of principal components, unweighted pair-group method with arithmetic mean dendrograms, fastStructure, and minimum spanning networks, and we obtained the index of association, diversity summary statistics, and genetic distance statistics values GST, G''ST, and Jost's D. Subpopulations of both species were mostly defined by their geographic origin indicating restricted dispersal of inoculum. Except for five isolates, the population structure of P. citrophthora (that is heterothallic and unlikely to reproduce sexually) was clonal to semi-clonal, with very little genetic diversity within and among subgroups. In contrast, the population structure of P. syringae was also clonal to semi-clonal, but isolates were placed into four main clusters of much higher diversity. Clonality in both species can be explained by a high level of asexual reproduction. The higher diversity in the homothallic P. syringae is likely due to commonly occurring sexual reproduction. One distinct cluster of P. syringae consisted solely of isolates from noncitrus hosts; therefore, the origin of P. syringae in citrus could not be resolved.

Regional Comparisons of Sensitivities of Phytophthora citrophthora and P. syringae Causing Citrus Brown Rot in California to Four New and Two Older Fungicides.

Isolates of the citrus brown rot pathogens Phytophthora citrophthora and P. syringae from the Inland Empire (IE) and Ventura Co. (VE) regions of southern California were evaluated for their sensitivity to ethaboxam, fluopicolide, mandipropamid, and oxathiapiprolin, and the previously published baselines that were generated for Central Valley (CV) isolates of California were expanded. Fungicides were generally more toxic to CV isolates of both species for all four fungicides. Specific differences were found in the toxicity of ethaboxam to P. syringae where CV isolates on average were 6.8 or 8.2 times more sensitive than those from the VE or IE regions, respectively. Based on the grouping of isolates in an unweighted pair-group method with arithmetic mean (UPGMA) dendrogram, as well as fastStructure analyses and plotting of principal component analyses (PCAs), differences in ethaboxam sensitivity could be related to differences in genetic background of the isolates. Isolates of P. citrophthora from the IE and VE had slightly reduced (i.e., 1.5×) sensitivity to mandipropamid as compared with isolates from the CV and were found on distinct branches in the UPGMA dendrogram. Differences in genetic background of less sensitive isolates within each species indicate that these two phenotypes emerged multiple times independently. IE and VE isolates of both species were sensitive to mefenoxam. Moderate resistance to potassium phosphite (EC50 values of 25 to 75 µg/ml) was present in IE and VE isolates of P. syringae, whereas some IE isolates of P. citrophthora were considered resistant with EC50 values of up to 113.69 µg/ml. Resistance to potassium phosphite did not relate to distinct genotypes.

A Novel IncX Plasmid Mediates High-Level Oxytetracycline and Streptomycin Resistance in Erwinia amylovora from Commercial Pear Orchards in California.

Isolates of the fire blight pathogen Erwinia amylovora with high-level resistance to oxytetracycline (minimal inhibitory concentration [MIC] > 100 µg/ml) and to streptomycin (MIC > 100 µg/ml) were recovered from four commercial pear orchards in California between 2018 and 2020. The two representative oxytetracycline- and streptomycin-resistant (OxyTcR-SmR) strains 32-10 and 33-1 were as virulent as the antibiotic susceptible strain 13-1 in causing blossom blight of pear and were recovered more than 50% of the time 7 days after co-inoculation to pear flowers with strain 13-1. In the field, inoculation of strain 32-10 to pear flowers that were pretreated with oxytetracycline at 200 µg/ml did not reduce disease compared with an untreated control. Four OxyTcR-SmR strains were subjected to draft genome sequencing to identify the genetic determinants of antibiotic resistance and their location. A 43.6-kb IncX plasmid, designated pX11-7, was detected in each of the four strains, and this plasmid encoded the tetracycline-resistance gene tetB and the streptomycin-resistance gene pair strAB within a large putatively mobile genetic element consisting of the transposon Tn10 that had inserted within the streptomycin-resistance transposon Tn6082. We also determined that pX11-7 was conjugative and was transferred at a rate that was 104 to 105 higher into an E. amylovora strain isolated in California compared with an E. amylovora strain that was isolated in Michigan. The occurrence of high levels of resistance to both oxytetracycline and streptomycin in E. amylovora strains from commercial pear orchards in California significantly limits the options for blossom blight management in these locations.

New Preharvest Treatments and Strategies in Managing Phytophthora Brown Rot of Citrus in California.

Brown rot caused by Phytophthora citrophthora, P. nicotianae, P. syringae, and P. hibernalis is an important fruit disease of citrus in California, and the latter two species are quarantine pathogens in some important export markets. The newly registered fungicides oxathiapiprolin (OXA) and mandipropamid (MAN), as well as a premixture of the two (MAN + OXA) were compared with standard fixed copper and potassium phosphite (KPO3) treatments (all with different modes of action) under field conditions in two citrus production regions of California. Fruit were sampled periodically over 8 weeks after application in winter or spring seasons, inoculated with zoospores of P. citrophthora or P. syringae, and brown rot incidence was evaluated. Single applications with all fungicides significantly reduced brown rot incidence of fruit harvested after 8 weeks as compared with the control in seasons with different amounts of precipitation (i.e., 17.2 to 153.9 mm between application and the 8-week sampling). MAN and OXA were similarly or significantly more effective than copper or KPO3. Two applications done in November and January significantly improved the efficacy of KPO3 and copper when compared with a single application of each fungicide done in January. For MAN and OXA, however, a single application was similar in efficacy as two applications. Two-application rotations of MAN, OXA, MAN + OXA, or copper significantly reduced the disease incidence by >84% from the control for at least 8 weeks after the second application. Low-volume (935 liters/ha) applications of MAN, OXA, MAN + OXA, or KPO3, but not copper, were significantly more effective than industry standard high-volume (3,740 liters/ha) applications. Thus, our studies identified and supported registration of new preharvest fungicide treatments to manage brown rot of citrus that are highly effective and persistent, and we optimized treatment strategies. Additionally, rotational programs with fungicides with different modes of action will minimize resistance development in pathogen populations and extend the usage of these fungicides.

Root Absorption and Limited Mobility of Mandipropamid as Compared to Oxathiapiprolin and Mefenoxam After Soil Treatment of Citrus Plants for Managing Phytophthora Root Rot.

Phytophthora root rot can greatly impact citrus production worldwide, especially in newly established orchards by reducing crop yield and increasing the cost of disease management. Mandipropamid is an Oomycota fungicide that is currently registered as a soil treatment for citrus nursery container plants to manage Phytophthora root rot. In this study, we investigated the uptake of mandipropamid into citrus roots and its translocation to stems and leaves after soil application and evaluated its mobility in roots as compared to oxathiapiprolin and mefenoxam using split-root potted plants and trees in the field. A bioassay and liquid chromatography-tandem mass spectrometry were used to detect and quantify fungicides in citrus tissues, and overall, similar results were obtained using the two methods. When applied to the soil of potted, 6- to 7-month-old citrus plants using labeled rates, the majority of mandipropamid was found in root tissues (4.9 to 18.1 µg/g), but small amounts were also present in stems (0.18 to 0.32 µg/g) and leaves (0.03 to 0.22 µg/g). There was no significant increase in concentrations in all three tissues between 1 and 4 weeks after application. Concentrations in all tissues exceeded established EC50 values for mycelial growth inhibition of P. citrophthora and P. nicotianae, the main citrus root rot pathogens in California. In a split-root study where the root systems of single plants were separated, no basipetal phloem-based mobility of mandipropamid or oxathiapiprolin was observed, but relative uptake into roots was higher for mandipropamid. In contrast, low amounts of mefenoxam were also present in roots in the untreated soil. Similar results were obtained in a field study where part of the root system was treated, and fungicides were extracted from nontreated roots. All three fungicides persisted inside roots over the 8-week period of this study. Uptake and persistence inside roots, as well as the previously reported high efficacy against citrus root rot in greenhouse and field studies support the use of mandipropamid in citrus nurseries and potentially in the orchard.

Mutations in Sdh Gene Subunits Confer Different Cross-Resistance Patterns to SDHI Fungicides in Alternaria alternata Causing Alternaria Leaf Spot of Almond in California.

Alternaria leaf spot caused by Alternaria alternata and A. arborescens is a common disease of almond in California. Succinate dehydrogenase inhibitors (SDHIs) are widely used for its management; however, we observed reduced performance of SDHI fungicides at some field sites. Thus, we evaluated the sensitivity to boscalid of 520 isolates of the main pathogen A. alternata collected from major production areas between 2006 and 2019, and also evaluated the sensitivity of a subset of 204 isolates to six members of the SDHIs belonging to six subgroups. Additionally, 97 isolates (14 sensitive and 83 with reduced sensitivity) of the 204 were used to determine the molecular mechanisms of resistance. A wide range of in vitro concentrations to effectively inhibit mycelial growth by 50% (EC50 values) was determined for each fungicide using the spiral gradient dilution method. Some isolates were highly resistant (EC50 values >10 µg/ml) to boscalid (a pyridine-carboxamide), pyraziflumid (a pyrazine-carboxamide), and fluxapyroxad (a pyrazole-4-carboxamide), but not to fluopyram (a pyridinyl-ethyl-benzamide), isofetamid (a phenyl-oxo-ethyl thiophene amide), and pydiflumetofen (a N-methoxy-(phenyl-ethyl)-pyrazole-carboxamide). There was no strong cross resistance among the fungicides tested, including for the two pyrazole-4-carboxamides fluxapyroxad and penthiopyrad (tested for 33 of the 204 isolates). The comparison of EC50 values for fluopyram and isofetamid resulted in the highest coefficient of determination (R2 = 0.582) among 10 pairwise comparisons between subgroups. Sequence analyses of the 97 isolates revealed five mutations in SdhB, SdhC, or SdhD subunits of the Sdh target gene among 73 isolates with reduced sensitivity to at least one SDHI. No mutations were detected in the 14 sensitive isolates and in 10 of the 83 isolates with reduced sensitivity. The most common mutation (59 isolates) was H134R in SdhC. Other mutations included H277Y (eight isolates) and H277L (two isolates) in SdhB, as well as G79R (two isolates) and S135R (two isolates) in SdhC. Mutations H277Y in SdhB and S135R in SdhC were only present in isolates collected in 2012 or earlier. Both conferred mostly high levels of resistance to boscalid and also reduced sensitivity to pyraziflumid, fluxapyroxad, and isofetamid with intermediate EC50 levels. Mutations H277L in SdhB, as well as H134R and G79R in SdhC, found in isolates obtained after 2012 had very similar resistance phenotypes with different levels of resistance to boscalid, pyraziflumid, and fluxapyroxad, whereas sensitivity to fluopyram, isofetamid, and pydiflumetofen was mostly less affected. Our data for SDHI fungicides do not support the classical concept of positive cross resistance within a single mode of action. Because some mutations conferred resistance to multiple SDHI subgroups, however, resistance management needs to consider all SDHIs as a homogenous group that should be mixed or rotated with other modes of action to delay development of resistance.

Natamycin, a Biofungicide for Managing Major Postharvest Fruit Decays of Citrus.

The antifungal polyene macrolide natamycin was evaluated as a postharvest biopesticide for citrus fruit. Aqueous spray applications with 1,000 µg/ml were moderately to highly effective against green mold incidence after inoculation but did not reduce sporulation of Penicillium digitatum on infected fruit. Treatments with natamycin were significantly more effective against green mold on grapefruit and lemon than on orange and mandarin, with 92.9, 88.5, 57.5, and 60.9% reductions in decay, respectively, as compared with the control. The biofungicide was compatible with a storage fruit coating but was less effective when applied in a packing coating. However, when either fruit coating was applied following an aqueous natamycin treatment (i.e., staged applications), the incidence of decay was reduced to ≤10.7% as compared with the untreated control (with 81.9%). The incidence of sour rot of lemon and mandarin was also significantly reduced from the untreated control by natamycin (1,000 µg/ml) but propiconazole (540 µg/ml) and propiconazole + natamycin (540 + 500 µg/ml) mixtures generally were significantly more effective than natamycin alone when using a severe inoculation procedure. Experimental and commercial packingline studies demonstrated that natamycin-fludioxonil or natamycin-propiconazole mixtures applied in a storage fruit coating or as an aqueous flooder treatment were highly effective and typically resulted in a >85.0% reduction of green mold and sour rot. Resistance to natamycin has never been documented in filamentous fungi. Thus, the use of natamycin, in contrast to other registered postharvest fungicides for citrus, can be an antiresistance strategy and an effective treatment in mixtures with other fungicides for the management of major postharvest decays of citrus.

Resistance to Potassium Phosphite in Phytophthora Species Causing Citrus Brown Rot and Integrated Practices for Management of Resistant Isolates.

Phytophthora citrophthora, P. syringae, P. nicotianae, and less commonly P. hibernalis are causal agents of brown rot of citrus fruit in California. The chronic disease occurs during the winter season, requires annual management, and has limited California citrus exports because of quarantines in some markets. Potassium phosphite (KPO3) is registered as a pre- and postharvest fungicide in the United States to manage Phytophthora brown rot. We evaluated the in vitro toxicity of KPO3 to 65, 60, and 38 isolates of P. citrophthora, P. syringae, and P. nicotianae, respectively, that were obtained from major growing regions of California. Frequency distributions of effective concentrations to inhibit mycelial growth by 50% (EC50 values) were not normally distributed, with skewness values of 1.84, 1.60, and -0.51 for each species, respectively. Isolates considered sensitive (EC50 values <25 µg/ml), moderately resistant (EC50 values from 25 to 75 µg/ml), or resistant (EC50 values >75 µg/ml) were identified for each species. The majority of P. citrophthora (83.1%) and P. syringae (78.3%) isolates were sensitive, whereas most P. nicotianae isolates (86.8%) were moderately resistant or resistant. Resistance factors were calculated as 65, 19, and 10 for the three species, respectively. In preharvest field trials, KPO3 (2,280 g/ha) applications were not effective in reducing citrus brown rot incidence when orange fruit were inoculated with a resistant (EC50 = 161.9 µg/ml) isolate of P. citrophthora, demonstrating the potential for field resistance. Oxathiapiprolin (32.6 g/ha), however, was highly effective, indicating the absence of multidrug resistance. Postharvest treatments with KPO3 were only effective in reducing brown rot caused by the resistant isolate of P. citrophthora to a low incidence when high rates (8,000 µg/ml) were used in heated (54°C) applications. The sensitive and moderately resistant isolates were managed using rates of 4,000 µg/ml, but heated treatments at this rate were needed to reduce brown rot to commercially acceptable levels when decay was caused by a moderately resistant isolate.

Organic Acid Sanitizers for Natamycin and Other Fungicides in Recirculating Application Systems for Citrus Postharvest Decay Management.

Natamycin is a new postharvest biofungicide for citrus and some other fruit crops in the United States that can be effectively used in recycling drench or flooder treatments. These applications necessitate sanitation of the fungicide solution to ensure that it remains free from contamination by bacteria that are potentially human pathogens. During in vitro experiments, heated (48°C) citric acid (1,100 or 2,200 µg/ml) amended with sodium dodecylbenzenesulfonate (SDBS) (60 or 120 µg/ml, respectively) significantly reduced the viability of a nonpathogenic strain of Escherichia coli in natamycin solutions by >5 log10 compared with the control. During laboratory studies with Penicillium digitatum-inoculated lemon fruit, 1,000 µg/ml of natamycin mixed with 1,000 µg/ml of lactic acid or citric acid and with or without SDBS (55 µg/ml) effectively and significantly reduced green mold. Natamycin mixed with lactic acid at ≥2,000 µg/ml, however, caused fruit injury, resulting in browning and rind pitting. Natamycin was incompatible with peroxyacetic acid, resulting in reduced efficacy against green mold. Sodium hypochlorite mixed with natamycin lost its toxicity to E. coli; however, the performance of natamycin was not affected. With heated (average 49°C) drench treatments on an experimental packing line, natamycin (1,000 µg/ml), fludioxonil (300 µg/ml), or azoxystrobin (300 µg/ml) mixed with citric acid (1,000 µg/ml) and SDBS (55 µg/ml) were effective against green mold without fruit injury. At a pH between 3.6 and 3.8, citric acid-SDBS significantly reduced the viability of E. coli by approximately 4 log10 in mixtures with fludioxonil or azoxystrobin, but not with natamycin. However, natamycin at 1,000 µg/ml mixed with 2,000 µg/ml of citric acid and SDBS (55 µg/ml) significantly reduced E. coli counts by >4 log10 within 4 min when the pH was maintained between 3.0 and 3.3, and the efficacy of the fungicide was retained. The use of citric acid with a surfactant can be a viable alternative sanitation method for natamycin in citrus packinghouses utilizing heated recirculating fungicide systems.

Epidemiology and Management of Bacterial Spot of Almond Caused by Xanthomonas arboricola pv. pruni, a New Disease in California.

Bacterial spot caused by Xanthomonas arboricola pv. pruni was first detected on almond in California in 2013, and it is reported herein as a new disease in California based on fulfilling Koch's postulates and identification of the pathogen using species-specific PCR primers. Infected mummified fruit from the previous growing season and their peduncles were identified as primary overwintering sites of the bacterium on the tree. Twig cankers were not observed, and the pathogen was not recovered from dormant buds. Isolation from flowers and emerging leaves was only successful when they were collected within 20 cm of an infected, mummified fruit on the tree. Inoculation of flowers and immature fruit as well as immature and mature leaves resulted in disease development, indicating a long period of host susceptibility in the spring, but disease incidence was highest in fruit inoculations. In split-plot trials over 3 years, dormant applications in December or January with copper or copper-mancozeb significantly reduced the disease compared with untreated controls in seasons with high rainfall, but they had no effect in seasons with low rainfall. In-season applications of copper-mancozeb at petal fall or at full bloom and petal fall were also effective in reducing the disease. Phytotoxicity was observed after repeated applications of copper bactericides, especially in low-rainfall seasons. Dormant and in-season treatments of copper-mancozeb mixtures integrated with removal of mummified fruit are currently the best management strategies for bacterial spot of almond in California.

Mobility of Oxathiapiprolin and Mefenoxam in Citrus Seedlings After Root Application and Implications for Managing Phytophthora Root Rot.

Oxathiapiprolin is highly effective in the management of Phytophthora root rot of citrus; however, its uptake into plants after soil application is not known. This was investigated and compared with mefenoxam using potted citrus seedlings sampled 7, 10, 13, and 16 days after soil treatments. Bioassays and high-performance liquid chromatography tandem mass spectroscopy (HPLC-MS/MS) were used to quantify fungicide amounts in plant extracts. Distinct inhibition zones of mycelial growth of Phytophthora citrophthora were observed in bioassays when root, stem, or leaf extracts were added to filter paper disks on agar plates. Based on the two quantification methods, concentrations of both fungicides in the three tissue types and at all sampling times were above the mean effective concentration that provides 50% growth reduction values of the baseline sensitivities. Relative concentrations at the four sampling times sometimes varied between the two methods but, for both methods, concentrations of oxathiapiprolin were significantly higher in roots and leaves as compared with stems 10 days after treatment and statistically similar in the three tissues after 7 days. For mefenoxam, concentrations significantly increased in roots between 7 and 16 days after treatment and were significantly the highest in roots as compared with stems or leaves 16 days after treatment. Regressions of oxathiapiprolin and mefenoxam concentrations using HPLC-MS/MS on those calculated from bioassay standard curves indicated that the bioassays overestimated fungicide amounts in the extracts. The bioassay, however, can be considered an alternative option comparable with costly residue analyses in fungicide mobility studies in plants. Uptake of oxathiapiprolin at sufficient but low concentrations into plant roots provides an explanation for its long-lasting high activity in the management of Phytophthora root rot.

Evaluation of New Oomycota Fungicides for Management of Phytophthora Root Rot of Citrus in California.

Phytophthora root rot, caused by several species of Phytophthora, is an important disease of citrus in California and other growing regions. For chemical management, mefenoxam and potassium phosphite have been available for many years, and resistance in Phytophthora spp. has been reported for both compounds. We evaluated the efficacy of the new Oomycota fungicides ethaboxam, fluopicolide, mandipropamid, and oxathiapiprolin, each with a different mode of action, against Phytophthora root rot of citrus in field and greenhouse studies. Root balls of navel orange trees on 'Carrizo citrange' rootstock were inoculated with P. nicotianae at planting in the field in fall 2013. Applications with 11 fungicide treatments were made 5 weeks after planting, in spring and fall 2014, and in spring 2015. Feeder roots and adjacent soil were collected before or after application. All of the new fungicides significantly reduced root rot incidence and Phytophthora soil populations to very low levels as compared with the control starting after the first application. Mefenoxam was only effective when a high label rate was used in the fourth application. Selected treatments also increased tree canopy size, trunk diameter, and fruit yield as compared with the control. A rate comparison with the four new fungicides was initiated in summer 2016 in another field trial using navel orange trees inoculated with P. citrophthora. Minimum effective rates to reduce Phytophthora root rot incidence and pathogen soil populations were determined after one and two applications in fall 2016 and summer 2017, respectively. Greenhouse studies confirmed the efficacy of the new fungicides. Based in part on our studies, fluopicolide recently received a federal and oxathiapiprolin a full registration for use on citrus, and registrations for ethaboxam and mandipropamid have been requested. These new compounds will provide highly effective treatment options and resistance management strategies using rotation and mixture programs for the control of Phytophthora root rot of citrus.

Temporal Occurrence and Niche Preferences of Phytophthora spp. Causing Brown Rot of Citrus in the Central Valley of California.

Brown rot of citrus fruit is caused by several species of Phytophthora and is currently of serious concern for the California citrus industry. Two species, Phytophthora syringae and P. hibernalis, are quarantine pathogens in China, a major export market for California citrus. To maintain trade and estimate the risk of exporting a quarantine pathogen, the distribution and frequency of Phytophthora spp. causing brown rot of orange in major growing areas of California was investigated. Symptomatic fruit were collected from navel (winter to late spring) and Valencia (late spring to summer) orange orchards from 2013 to 2015. Species identification of isolates was based on morphological characteristics, random amplified polymorphic DNA banding patterns, and sequencing of the internal transcribed spacer and the partial cox2/spacer/cox1 regions from axenic cultures, or directly on DNA from fruit tissue using a multiplex TaqMan quantitative polymerase chain reaction assay. In winter samplings, the incidence of P. syringae based on the number of fruit with Phytophthora spp. detection ranged from 73.6 to 96.1% for the two counties surveyed. The remaining isolates were identified as P. citrophthora. In late spring or summer, only P. citrophthora was recovered. P. hibernalis and P. nicotianae were not detected in any fruit with brown rot symptoms. These results indicate that P. syringae is currently an important brown rot pathogen of citrus fruit in California during the cooler seasons of the year. In winter 2016 and 2017, P. syringae was recovered by pear baiting at a high incidence from leaf litter and from a small number of rhizosphere soil or root samples but not from living leaves on the tree. In contrast, P. citrophthora was rarely found in leaf litter but was commonly detected in the rhizosphere. Thus, leaf litter is a major inoculum source for P. syringae and this species occupies a distinct ecological niche.

Baseline Sensitivities of New Fungicides and Their Toxicity to Selected Life Stages of Phytophthora Species from Citrus in California.

Phytophthora citrophthora, P. syringae, P. nicotianae, and P. hibernalis are important pathogens of citrus in California but few chemical treatments are currently available. In vitro toxicities of four new fungicides to isolates of Phytophthora spp. from California were determined. Mean effective concentration values to inhibit mycelial growth by 50% for ethaboxam, fluopicolide, mandipropamid, oxathiapiprolin, and mefenoxam were 0.068, 0.04, 0.004, 0.0003, and 0.039 µg/ml, respectively, for 62 isolates of P. citrophthora; 0.005, 0.045, 0.003, 0.0001, and 0.008 µg/ml, respectively, for 71 isolates of P. syringae; 0.016, 0.057, 0.005, 0.0005, and 0.183 µg/ml, respectively, for 31 isolates of P. nicotianae; and 0.030, 0.018, 0.005, <0.0003, and ≤0.001 µg/ml, respectively, for two isolates of P. hibernalis. Mean values for ≥90% inhibition of sporangia formation of four isolates of P. citrophthora were 0.1, 0.28, 0.026, 0.005, and 55 µg/ml for ethaboxam, fluopicolide, mandipropamid, oxathiapiprolin, and mefenoxam, respectively. Zoospore cyst germination of P. citrophthora was most inhibited by oxathiapiprolin and mandipropamid. Chlamydospore formation of P. nicotianae was most sensitive to oxathiapiprolin, with a mean ≥90% reduction (EC>90) of 0.002 µg/ml, moderately sensitive to mandipropamid (EC>90 = 0.2 µg/ml) and mefenoxam (EC>90 = 0.6 µg/ml), and least sensitive to ethaboxam and fluopicolide (EC>90 = 1 µg/ml). Oospore formation of P. nicotianae was inhibited by ≥90% using oxathiapiprolin at 0.0004 µg/ml, mandipropamid at 0.02 µg/ml, ethaboxam at 0.1 µg/ml, or fluopicolide at 0.4 µg/ml, whereas 62% inhibition was obtained by mefenoxam at 40 µg/ml.

Characterization of Streptomycin Resistance in Isolates of Erwinia amylovora in California.

In surveys from 2006 to 2014, streptomycin resistance in Erwinia amylovora from pear-growing areas in California declined from very high incidence in 2006 and 2007 to very low incidence in 2013 and 2014. The majority of resistant strains were designated as moderately resistant-low (MR-L), and were almost exclusively found in Sacramento County, whereas highly resistant (HR) strains were only recovered in Sutter-Yuba and San Joaquin counties. Resistance of HR strains was associated with a mutation in codon 43 of the chromosomal rpsL gene that results in a change from lysine to arginine, the same mutation that was originally reported for resistant strains from California in the mid-1970s. MR-L strains were found to harbor the strA-strB streptomycin resistance genes on transposon Tn5393a. This transposon lacks insertion sequence IS1133 that provides a promoter for efficient expression of strA-strB, resulting in lower minimum inhibitory concentrations of MR-L strains compared with those from other locations that harbor strA-strB on Tn5393::IS1133. In contrast to previously described plasmid-mediated resistance where Tn5393 is inserted in pEa34, or pEA29, Tn5393a in MR-L strains was located on plasmid pEU30. This plasmid was first described in E. amylovora from the western United States but was not associated with streptomycin resistance determinants previously. We hypothesize that Tn5393a was introduced into an E. amylovora strain carrying pEU30 and transposed into that plasmid. This hypothesis was supported by clustered regularly interspaced short palindromic repeat (CRISPR) sequence analysis that showed that two MR-L strains share the same CRISPR1 pattern as a streptomycin-sensitive strain. With current low resistance levels in California growing regions, streptomycin could be successfully used again, but applications per season should be limited and the antibiotic should be mixed and rotated with different modes of action.

Pythium brassicum sp. nov.: A Novel Plant Family-Specific Root Pathogen.

Roots of stunted broccoli plants (Brassica oleracea) from the Palo Verde Valley, CA, were observed at various stages of decay. A species of Pythium with large spiny oogonia was microscopically observed and consistently isolated from decayed roots. Isolates produced spherical, intercalary sporangia (average 34.5 µm in diameter) and aplerotic oospores (average 37.3 µm in diameter) in oogonia (average 47.4 µm in diameter) bearing numerous conical spines (average 8.5 µm in height and 5.5 µm basal width) with blunt apices. A representative broccoli isolate (P1) had a 99% internal transcribed spacer (ITS) sequence similarity with Pythium jasmonium (nom. inval., GenBank AF216654.1), a species which has not been formally described. Three other accessions in GenBank also carry the specific epithet of P. jasmonium and were originally isolated from diseased plants in the family Brassicaceae. In our study, these isolates were pathogenic on broccoli and morphologically similar to P1. P1 was pathogenic to 10 cultivated and 12 wild plants in the family Brassicaceae but not to 18 species of cultivated plants belonging to nine other plant families. Mycelial growth of our isolates occurred between <10 and 35°C, with an optimum of 25°C (maximum growth rate 25 mm/day). Our broccoli isolates are related to other species in subclade B of clade J. Members of subclade B include P. mastophorum, P. uncinulatum, P. buismaniae, P. polymastum, and P. megalacanthum. However, the broccoli isolates, in addition to all those in GenBank that carry the specific epithet of P. jasmonium, possess unique ITS, ß-tubulin, and cox1 sequences that are sufficiently different from other species in subclade B to justify status as a new species. We propose that isolates previously designated as P. jasmonium (nom. inval.) be renamed as P. brassicum sp. nov. based on their apparent plant family-specific pathogenicity.

Optimizing Efficacy of New Postharvest Fungicides and Evaluation of Sanitizing Agents for Managing Citrus Green Mold.

Three new fungicides, azoxystrobin, fludioxonil, and pyrimethanil, that belong to different chemical classes are highly effective in managing citrus green mold and are being registered for postharvest use in the United States. Recirculating in-line drenches provided a significantly improved efficacy compared with standard low-volume spray applications. To prevent pathogen contamination of drench solutions, two oxidizing disinfectants, sodium hypochlorite and hydrogen peroxide/peroxyacetic acid (HPPA) solutions, were evaluated. Inhibition of conidial germination of Penicillium digitatum was dependent on the pH of the solution and the exposure time for each sanitizing agent. Chlorine (50 mg/liter) and HPPA (2,700 mg/liter) effectively inhibited germination in 40- and 240-s exposures, respectively, at pH 7. All fungicides tested were compatible and effective with HPPA, whereas fludioxonil, azoxystrobin, and thiabendazole, but not imazalil and pyrimethanil, were compatible with chlorine. In laboratory studies, sodium bicarbonate (SBC, 3%) significantly increased the efficacy of the three fungicides (250 mg/liter) and had no adverse effect on their stability in aqueous solutions. Fludioxonil (300 mg/liter)-SBC mixtures were still highly effective when applied 24 h after fruit inoculation. In experimental packingline studies, SBC or SBC-chlorine improved the efficacy of fludioxonil, whereas azoxystrobin was effective with and without these additives. Heating of drench solutions of fludioxonil (300 mg/liter) to 50°C did not improve decay control. In conclusion, in-line recirculating drench applications and fungicide-sanitizer-SBC mixtures significantly increased fungicide efficacy and provide an integrated approach for optimizing fungicide efficacy. These strategies also should minimize the selection for resistant isolates of the pathogen.

Baseline Sensitivities for New Postharvest Fungicides Against Penicillium spp. on Citrus and Multiple Resistance Evaluations in P. digitatum.

For the first time in over 25 years, three new fungicides (azoxystrobin, fludioxonil, and pyrimethanil), all belonging to different chemical classes, are being registered for postharvest use against Penicillium decays of citrus fruit in the United States. Baseline sensitivities of Penicillium digitatum and P. italicum were developed using isolates collected before the commercial use of these new fungicides. In a comparison of methods, EC50 values obtained using the spiral gradient dilution method were very similar to those obtained using traditional agar dilutions of fungicides. For azoxystrobin, the addition of salicylhydroxamic acid (SHAM) did not significantly affect EC50 values for mycelial growth of both species. In additional studies on conidial germination of P. digitatum, SHAM significantly reduced EC50 values for azoxystrobin. For pyrimethanil, the mean EC50 value for mycelial growth obtained using a minimum growth medium for anilinopyrimidine fungicides was significantly lower but comparable to values obtained when using potato dextrose agar . For mycelial growth of P. digitatum, mean EC50 values were 0.014, 0.025, and 0.313 µg/ml, whereas for conidial germination, they were 0.074, 0.163, and 1.195 µg/ml for azoxystrobin, fludioxonil, and pyrimethanil, respectively. For P. italicum, mean EC50 values for mycelial growth for fludioxonil and pyrimethanil were 0.005 and 0.040 µg/ml, respectively. For azoxystrobin, the mean EC50 value for mycelial growth for 33 isolates was 0.029 µg/ml. Four isolates had EC50 values ≥0.772 µg/ml and were considered part of a resistant subpopulation. Multiple resistance between the older and new postharvest fungicide classes on citrus was not detected in P. digitatum, and all isolates that were sensitive or resistant to imazalil or thiabendazole were sensitive to the new compounds. This information is important for monitoring populations of P. digitatum, where resistance against the older fungicides has commonly developed.

Natamycin, a New Biofungicide for Managing Crown Rot of Strawberry Caused by QoI-Resistant Colletotrichum acutatum.

Anthracnose crown rot of strawberry, caused by Colletotrichum acutatum, is an important disease affecting California nursery and fruit production. Preplant dip treatments of transplants with fludioxonil-cyprodinil or azoxystrobin are industry standards for managing the disease and have been used extensively. Following reports of reduced efficacy of azoxystrobin in the field, high levels of quinone outside inhibitor (QoI) resistance were detected in California isolates of the pathogen. Resistance was associated with the G143A mutation in the cytochrome b gene, similar to a previous report from Florida, and there were no detected fitness penalties in pathogenicity or virulence. Therefore, several alternative fungicides were investigated in laboratory and field studies. Subsequently, the new biofungicide natamycin was identified. Baseline sensitivities of 74 isolates of C. acutatum to natamycin were determined to be unimodal, with a range from 0.526 to 1.996 µg/ml (mean 0.973 µg/ml). Although this toxicity was considerably lower than that of azoxystrobin (using sensitive isolates), fludioxonil, or cyprodinil, dip treatments of transplants with natamycin (at 500 or 1000 mg/liter) were highly effective. Disease severity and plant mortality in field studies with inoculated transplants were reduced to similarly low levels as treatments containing fludioxonil, whereas azoxystrobin failed in inoculations with QoI-resistant isolates of C. acutatum. Fruit yield was also significantly increased by natamycin as compared with the inoculated control. Differences in disease susceptibility were observed among cultivars evaluated, with Monterey and Portola more susceptible than Fronteras. Natamycin has a unique mode of action that is different from other fungicides registered on strawberry and, based on this research, was registered in the United States as a preplant, biofungicide dip treatment of strawberry transplants for management of anthracnose crown rot.

Comparative Efficacy of the New Postharvest Fungicides Azoxystrobin, Fludioxonil, and Pyrimethanil for Managing Citrus Green Mold.

Three new fungicides (i.e., azoxystrobin, fludioxonil, and pyrimethanil) are currently being introduced for postharvest management of citrus green mold in the United States. The effectiveness of each fungicide was evaluated when applied alone (at 1,000 to 1,200 mg/liter) or in mixtures (at 500 mg/liter each component) to lemon fruit that were wound-inoculated with imazalil/thiabendazole (TBZ)-sensitive or -resistant isolates of Penicillium digitatum. In laboratory studies when aqueous fungicide solutions were applied 9 to 21 h after inoculation, pyrimethanil showed the highest level of green mold control. The efficacy of fludioxonil and azoxystrobin was very high at the early timings, but decreased as time after inoculation increased. Differences in fungicide performance were not due to multiple fungicide resistance, but more likely due to differences in fungicide mobility in fruit tissue. Azoxystrobin-fludioxonil mixtures were significantly more effective when compared to single-fungicide treatments. Mixtures of imazalil with pyrimethanil were the most effective in controlling decay. The efficacy of all fungicides was significantly lower when mixed into a packing fruit coating as compared to aqueous or storage fruit coating applications. In laboratory and packingline studies, the lowest incidence of green mold decay was obtained when azoxystrobin-fludioxonil and imazalil-pyrimethanil were applied as aqueous solutions that were followed by a fruit coating. Among the new fungicides, azoxystrobin and fludioxonil applied in water or storage fruit coating, respectively, provided the best anti-sporulation activity. Storage fruit coating improved the activity of both fungicides. Pyrimethanil was the least effective fungicide in suppressing sporulation of the pathogen on decaying fruit. Overall, among the mixtures, azoxystrobin-fludioxonil and TBZ-fludioxonil had high anti-sporulation activity in aqueous and storage fruit coating applications. New integrated management programs should be based on monitoring of fungicide sensitivities in pathogen populations, rotating mixtures of products with different modes of action, and using appropriate fungicide application strategies.