Alternaria Late Blight Research Articles

Efficacy of fungicides against Alternaria late blight and their effects on resistance levels

Efficacy of fungicides against Alternaria late blight and their effects on resistance levels

Frequency of Alternaria Genotypes Resistant to SDHI Fungicides in California Pistachio Orchards Determined by Real-Time PCR.

Real-time PCR methods were developed to quantify the frequency of SDHC-H134R and SDHB-H277Y mutants associated with succinate dehydrogenase inhibitors (SDHI) resistance in Alternaria populations from pistachio. The linearity of the standard curves demonstrated the applicability in the quantification of the assays. The accuracy and reliability of the qPCR protocols to determine the frequency of mutants in real samples were corroborated. Orchards visibly affected by Alternaria late blight were sampled. The frequency of mutants was determined using the qPCR assays, while the frequency of resistant phenotypes was determined using a single discriminatory dose. The statistical analysis showed that the frequencies of the mutation SDHC-H134R determined with the qPCR assay were highly correlated with those estimated with the conventional method. The survey also evidenced that resistance to boscalid is still widespread in California. Results also indicated the possible contribution of other mutations to SDHI resistance. Our results confirmed the prevalence of SDHC-H134R mutants and the occurrence of mutation SDHB-H277Y at low frequencies. The real-time PCR methods developed in this study were able to detect differences in the frequencies of resistant mutants caused by the use of chemical fungicides. Finally, the effects of two fungicide programs on the frequency of mutants resistant to SDHI and quinone outside inhibitors fungicides were studied using qPCR assays. The experiments demonstrated that the use of anilinopyrimidine and demethylation inhibitors fungicides in the same program reduced the frequency of these mutations in Alternaria populations. The qPCR methods developed and used in this study can be used to track resistance levels in the pistachio orchards on a large scale.

Occurrence and Extent of Boscalid Resistance in Populations of Alternaria alternata from California Pistachio Orchards.

Alternaria late blight (ALB) caused by Alternaria spp. is an annual disease problem in California pistachio and requires repeated applications of fungicides to prevent significant losses of pistachio foliage and nut quality. From 2003 onward, the succinate dehydrogenase inhibiting fungicide boscalid has played a key role in ALB management. The development of boscalid resistance in A. alternata populations was monitored from 2005 to 2012 in pistachio producing areas in California. A total of 1,765 single-spore isolates, collected from commercial and experimental pistachio orchards with or without a history of boscalid exposure, were tested in a radial growth assay in agar media amended with the discriminatory dose of 10 µg/ml of boscalid. The frequency of boscalid-resistant isolates in 2005 was 12% but increased significantly and remained stable toward the end of the survey period. Most of the resistant isolates exhibited a high level of resistance (R) to boscalid with percent of mycelial growth inhibition (PGI) values between 0 and 50%, whereas significantly fewer isolates had an intermediate level (IR) of resistance (50 < PGI < 75%). The frequency of sensitive (S) isolates (75 < PGI < 100%) was generally the highest in orchards with no history of boscalid usage, whereas mean incidences of boscalid-resistant populations of Alternaria were 81, 92.4, 80.2, and 98%, in 2006, 2007, 2011, and 2012, respectively, in orchards that received a high number (at least three per season) of boscalid spray applications. In comparison, none to relatively low frequencies (0 to 12%) of resistance were observed in populations with no or limited exposure to Pristine, suggesting an air-movement of resistant spores through wind from treated to nontreated areas. In 2012, boscalid-resistant isolates were found practically in every sampled location in all counties, with the orchards in Fresno, Madera, Tulare, and King Counties being the locations with the highest frequencies of resistance (100%). Monitoring of A. alternata AaSDHB, AaSDHC, and AaSDHD mutations in 286 boscalid-resistant phenotypes identified 11 mutations, leading to amino acid substitutions in AaSDHB (seven mutations: H277Y/R/L, P230A/R, N235D/T), AaSDHC (one mutation: H134R), and AaSDHD (three mutations: D123E, H133R/P), with AaSDHB mutations being the most prevalent (80%) ones throughout the survey period. The majority of isolates carrying these mutations exhibited the R phenotype toward boscalid. The increased prevalence of boscalid resistance in populations of A. alternata is a likely contributing factor to the inability of pistachio farmers to successfully control ALB with Pristine. Other factors implicated in the rapid and widespread occurrence of A. alternata boscalid-resistant populations in California pistachios are further discussed.

Effect of the Presowing Treatment of Potato Tubers with Film Coating Containing Metal Nanoparticles on Pathologies and Yield Production

A preparation based on iron, zinc, copper, and molybdenum nanoparticles (NPs) in a polymer film formed from a mixture of Na-carboxymethylcellulose and polyethylene glycol-400 was developed for presowing pretreatment of potato tubers. Based on laboratory tests, the optimal concentrations and ratio of Fe : Zn : Cu : Mo nanoparticles were determined as 10–6 : 10–2 : 10–8 : 10–8 (%). Field tests in the integrated system of potato protection showed that the presowing treatment of potato tubers with nanoparticles Fe : Zn : Cu : Mo in the polymer film reduced the prevalence and degree of development of Alternaria blight (2.2 and 2.4 times) and late blight of potato (3.4 and 4.2 times) during the growing season and led to an increase in yield of marketable products by 11.7% compared to the control.

Using Allele-specific PCR for detecting multiple amino acid substitutions associated with SDHI resistance in Alternaria alternata causing Alternaria late blight in pistachio.

This study provides a rapid and cost-effective way to identify five different genotypes associated with Alternaria alternata resistance to succinate dehydrogenase inhibitors fungicides, used to control Alternaria late blight in pistachio. With the allele-specific method developed here, users will be able to identify genotypes with nucleotide substitutions which lead to a reduced sensitivity or resistance selection using a one-step PCR assay, without the use of restriction enzymes which elevates the reaction costs and the chance for errors. In addition, this study formally includes the identification and sequence accession for SdhB-H277L and SdhC-S135R amino acid substitution in A. alternata sampled from California and Arizona pistachio orchards.

Molecular and pathogenic characterization of Iranian isolates associated with leaf spot disease of potato

Received: 2017-10-05 | Accepted: 2018-01-23 | Available online: 2018-03-31 https://doi.org/10.15414/afz.2018.21.01.01-05 Alternaria alternata (Fr.) Keissler is one of the main causal agents of leaf spot on potato in Iran and worldwide. In this study, random amplified polymorphic DNA (RAPD) and pathogenicity assay were employed to analyze 28 A. alternata isolates obtained from potato plants. The isolates were collected from main potato growing regions of Iran, including Ardebil, Hamedan, Isfahan and Fars provinces. Cluster analysis of genotypes produced by RAPD marker, using UPGMA method indicated that the isolates have been clustered into different groups with no correlation to geographical origins of the isolates. Pathogenicity assay indicated that all A. alternata isolates were pathogenic on potato; however, virulence variability was observed among the isolates. The findings revealed that because of extant diversity in pathogenicity and genetics of A. alternata isolates, a single isolate should not be used for evaluating resistance of potato. Keywords: Alternaria alternata , pathogenicity, RAPD, Solanum tuberosum L. References ANDERSEN, B., HANSEN, M.E. and SMEDSGAARD, J. (2005) Automated and unbiased image analyses as tools in phenotypic classification of small-spored Alternaria spp. In Phytopathol, vol. 95, pp. 1021–1029. DROBY, S. et al. (1984) Pathogenicity of Alternaria alternata on potato in Israel. In Phytopathol, vol. 74, pp.537–542. KAKVAN, N. et al. (2012) Study on pathogenic and genetic diversity of Alternaria alternata isolated from citrus hybrids of Iran, based on RAPD-PCR technique. In Eur J Exp Biol, vol. 2, no. 3, pp. 570–576. MAHMODI, F. et al. (2014) Genetic Diversity and Differentiation of Colletotrichum spp. Isolates Associated with Leguminosae Using Multigene Loci, RAPD and ISSR. In Plant Pathol J, vol. 30, no. 1, pp. 10–24. McDONALD BA and LINDE, C. (2002) Pathogen population genetics, evolutionary potential, and durable resistance. In Annu Rev Phytopathol, vol. 40, pp. 349–379. MEENA, RK., SHARMA, SS. and SINGH, S. (2015) Studies on variability in Alternaria alternata (Kessler) causing leaf blight of Isabgol (Plantago ovata). In SAARC J Agric, vol. 12, no. 2, pp. 63–70. MORRIS, P.F., CONNOLLY, M.S. and CLAIR, D.A. (2000) Genetic diversity of Alternaria alternata isolated from tomato in California assessed using RAPDs. In Mycol Res, vol. 104, no. 3, pp. 286–292. NASEHI, A. et al. (2014) Analysis of genetic and virulence variability of Stemphylium lycopersici associated with leaf spot of vegetable crops. In Eur J Plant Pathol, vol. 140, no. 2, pp. 261–273. NASR ESFAHANI, M. and ANSARIPOUR, B. (2006) Identification of common leaf blight diseases of potato in Fereidan. In Proceedings of the 17th Iranian Plant Protection Congress, Campus of College of Agriculture and Natural Resources, University of Tehran. Teheran: College of Agric Sci, p. 213. NATIONAL INSTITUTION OF AGRICULTURE BOTANY (NIAB) (1985) Diseases assessment manual for crop variety trials. Cambridge: UP. PRYOR B.M. and MICHAILIDES, T.J. (2002) Morphological, pathogenic, and molecular characterization of Alternaria isolates associated with Alternaria late blight of pistachio. In Phytopathol, vol. 92, no. 4, pp. 406–416. ROHLF, E.J. (1993) NTSYS-pc: Numerical taxonomy and multivariate analysis system, version 1.80. Setauket, New York: Applied Biostatistics Inc. ROTEM, J. (1994) The Genus Alternaria; Biology, Epidemiology, and Pathogenicity. Minnesota, USA: American Phytopathological Society Press. 326 p. TALBOT, N. (2001) Molecular and Cellular Biology of Filamentous Fungi. New York, USA: Oxford University Press. WAALS Van der, J. E. et al. (2003) Evalution of Plant-plus, a decision support system for control of early blight on potatoes in South Africa. In Crop Prot, vol. 22, pp. 821–828. WAALS Van der, J. E. et al. (2011) First report of Alternaria alternata causing leaf blight of potatoes in South Africa. In Plant Dis, vol. 95, no. 3, pp. 363–363. WEIR, T. L. et al. (1998) RAPD-PCR analysis of genetic variation among isolates of Alternaria solani and Alternaria alternata from potato and tomato. In Mycol, pp. 813–821.

Survey of the pathogen of Alternaria late blight reveals different levels of carboxamide fungicide resistance on the main pistachio producing regions of California

Alternaria late blight (ALB), caused mainly by the fungal pathogen Alternaria alternata, is an important pistachio disease that causes severe tree defoliation and fruit shell staining. Its control relies on multiple fungicide sprays, including carboxamide fungicides. In 2015, we surveyed 35 orchards representing nine pistachio producing counties of California to determine the current situation of Alternaria resistance to four widely used carboxamide fungicide active ingredients. This survey showed that isolates collected in the northern (Tehama, Glenn and Colusa counties) and southern (Tulare, Kings and Kern counties) Central Valley presented higher frequencies of carboxamide resistance than isolates collected from orchards in the central region (Fresno, Madera and Merced counties). The number of carboxamide usages in a year is the main factor determining elevated resistance. By extracting the A. alternata DNA and sequencing the carboxamide target genes, we evaluated the prevalence of specific molecular alterations (mutations) associated with carboxamide fungicide resistance. Finally, we identified cross-resistance patterns among different carboxamide fungicides, leading to recommendations about combinations to avoid.

Sensitivity and cross-resistance patterns to demethylation-inhibiting fungicides in California populations of Alternaria alternata pathogenic on pistachio

Alternaria late blight (ALB) caused by Alternaria spp. is an annual production concern for pistachio growers in California. In this study, isolates of Alternaria alternata collected from California pistachio orchards between 1998 and 2003 (population A, n = 46) and in 2010 (population B, n = 38) prior to the registration and use of sterol demethylation-inhibiting (DMI) fungicides for ALB control, were tested for their sensitivity to difenoconazole, propiconazole, and tebuconazole using an in vitro mycelial growth assay. The 50% effective dose (EC50) values for isolates in population A to difenoconazole, propiconazole, and tebuconazole, respectively, ranged from 0.02 to 0.82 μg/mL (mean = 0.17), 0.25–5.73 μg/mL (mean = 1.29), and 0.02–2.76 μg/mL (mean = 0.96), and from 0.02 to 0.93 μg/mL (mean = 0.21), 0.13–5.36 μg/mL (mean = 1.25), and 0.17–5.47 μg/mL (mean = 1.03) for those in population B. Among 65 isolates collected in 2012 from orchards with very limited exposure to difenoconazole, propiconazole or metconazole (population C, n = 65), the EC50 values for tebuconazole ranged from 0.02 to 2.99 μg/mL (mean = 0.51), while EC50 values for difenoconazole of 55 of these isolates varied from 0.01 to 0.44 μg/mL (mean = 0.06). The DMI-exposed population C was found to be on average two to four times less sensitive compared with the populations A and B. Pearson correlation analyses of EC50 values for the three fungicides showed significant positive correlations between the sensitivities of tebuconazole and propiconazole (P < 0.01, r = 0.32), tebuconazole and difenoconazole (P < 0.01, r = 0.26), and difenoconazole and propiconazole (P < 0.01, r = 0.40). Results indicate a clear difference in fungicide intrinsic activity, with difenoconazole being more active than the two other DMIs. Although wide variations in sensitivities for tebuconazole and propiconazole were observed in the non- and DMI-exposed Alternaria populations, efficacy of DMI products has been excellent against ALB. The data collected here will be crucial for the rational use of DMIs in fungicide spray programs for ALB management and serve as a reference to detect any shifts in A. alternata sensitivity to these fungicides in subsequent years as they become more frequently used in California pistachios.

Sensitivities of Baseline Isolates and Boscalid-Resistant Mutants of Alternaria alternata from Pistachio to Fluopyram, Penthiopyrad, and Fluxapyroxad.

Resistance of Alternaria alternata to boscalid, the first succinate dehydrogenase inhibitor (SDHI) fungicide labeled on pistachio, has become a common occurrence in California pistachio orchards and affects the performance of this fungicide. In this study, we established the baseline sensitivities of A. alternata to the new SDHIs fluopyram, fluxapyroxad, and penthiopyrad and assessed their cross resistance patterns with boscalid. Examination of the effective fungicide concentration that inhibits mycelial growth to 50% relative to the control (EC50) for 50 baseline isolates revealed that the majority were sensitive to boscalid, penthiopyrad, fluopyram, and fluxapyroxad. Analysis of EC50 values for boscalid for 117 A. alternata isolates originating from boscalid-exposed orchards showed that 44, 3, 1, and 69 isolates had sensitive, reduced sensitivity, moderately resistant, and highly resistant boscalid phenotypes, respectively. Molecular investigation of the occurrence of known SDH mutations showed that, among the 69 isolates highly resistant to boscalid, 44, 2, 14, and 1 isolates possessed the mutations leading to the H277Y, H277R, H134R, and H133R amino acid substitutions in AaSDHB, AaSDHB, AaSDHC, and AaSDHD subunits, respectively. Some SDHB or SDHC mutants displayed highly sensitive, sensitive, or reduced sensitivity phenotypes toward penthiopyrad or fluxapyroxad, whereas other had low, moderate, or high levels of resistance to these fungicides. In contrast, all the SDHB mutants were sensitive to fluopyram, while 10, 5, and 1 SDHC mutants had sensitive, reduced sensitivity, and moderately resistant fluopyram phenotypes, respectively. The SDHD mutant had reduced sensitivity to fluopyram and penthiopyrad but was highly resistant to fluxapyroxad. The discrepancies of cross-resistance patterns between SDHIs suggest that their binding sites in complex II may differ slightly and that additional mechanisms of resistance to these compounds are likely involved. Ultimately, the findings of this study should lead to the rational and sustained deployment of new SDHIs in Alternaria late blight spray programs.

Competitive Ability and Fitness of Alternaria alternata Isolates Resistant to QoI Fungicides.

Fungicides that act as quinone outside inhibitors (QoIs) constitute a fungicide group extensively used against Alternaria late blight of pistachio caused by Alternaria spp. However, developement of resistance to this fungicide class constitutes an important threat for the succesful control of the disease. This study was conducted to determine whether development of resistance to QoIs is associated with a fitness cost, by measuring several biological and epidemiological parameters and estimating the competitive ability in four QoI-resistant and four QoI-sensitive Alternaria alternata isolates. Fitness parameters measured were mycelial growth and spore production in vitro, disease latent period, aggressiveness, and spore production on detached pistachio leaves. The competitive ability of resistant isolates was assessed in coinoculation experiments with sensitive isolates on detached pistachio leaves, using a real-time polymerase chain reaction assay technique. Fitness parameters between grouped QoI-resistant and QoI-sensitive isolates were not significantly different (P = 0.13, 0.21, 0.31, and 0.27 for sporulation in vitro, mycelial growth, incubation period, and sporulation in vivo, respectively), while resistant isolates, as a group, showed a higher aggressiveness (P = 0.01) compared with the sensitive isolates. The data indicate that the resistant strains did not account for a fitness cost compared with the sensitive ones under the conditions of testing. The outcome of the competition experiments was isolate dependent. In two pairs, the resistance frequencies increased whereas, in the remaining two pairs of isolates, resistance frequency decreased, suggesting that the resistant isolates were competitive similarly to the sensitive isolates.

Resistance to Boscalid Fungicide in Alternaria alternata Isolates from Pistachio in California.

Boscalid is a new carboxamide fungicide recently introduced in a mixture with pyraclostrobin in the product Pristine for the control of Alternaria late blight of pistachio. In all, 108 isolates of Alternaria alternata were collected from pistachio orchards with (59 isolates) and without (49 isolates) prior exposure to boscalid. The sensitivity to boscalid was determined in conidial germination assays. The majority of isolates from two orchards without a prior history of boscalid usage had effective fungicide concentration to inhibit 50% of spore germination (EC50) values ranging from 0.089 to 3.435 μg/ml, and the mean EC50 was 1.515 μg/ml. Out of 59 isolates collected from an orchard with a history of boscalid usage, 52 isolates had EC50 values ranging from 0.055 to 4.222 μg/ml, and the mean EC50 was 1.214 μg/ml. However, in vitro tests for conidial germination and mycelial growth also revealed that seven A. alternata isolates, originating from the orchard exposed to boscalid were highly resistant (EC50 > 100 μg/ml) to this fungicide. Furthermore, in vitro tests showed no significant differences between wild-type and boscalid-resistant mutants in some fitness parameters such as spore germination, hyphal growth, sporulation, or virulence on pistachio leaves. Experiments on the stability of the boscalid-resistant phenotype showed no reduction of the resistance after the mutants were grown on fungicide-free medium. Preventative applications of a commercial formulation of boscalid (Endura) at a concentration which is effective against naturally sensitive isolates failed to control disease caused by the boscalid-resistant isolates in laboratory tests. To our knowledge, this is first report of field isolates of fungi resistant to boscalid.

Resistance to pyraclostrobin, boscalid and multiple resistance to Pristine® (pyraclostrobin + boscalid) fungicide in Alternaria alternata causing alternaria late blight of pistachios in California

Pristine® (pyraclostrobin + boscalid) is a fungicide registered for the control of alternaria late blight in pistachio. A total of 95 isolates of Alternaria alternata collected from orchards with and without a prior history of Pristine® sprays were tested for their sensitivity towards pyraclostrobin, boscalid and Pristine® in conidial germination assays. The EC50 values for 35 isolates from orchards without Pristine® sprays ranged from 0·09 to 3·14 µg mL−1 and &lt; 0·01 to 2·04 µg mL−1 for boscalid and Pristine®, respectively. For pyraclostrobin, 27 isolates had EC50 &lt; 0·01 µg mL−1 and six had low resistance (mean EC50 value = 4·71 µg mL−1). Only one isolate was resistant to all three fungicides tested, with EC50 &gt; 100 µg mL−1. Among 59 isolates from the orchard with a history of Pristine® sprays, 56 were resistant to pyraclostrobin; only two were sensitive (EC50 &lt; 0·01 µg mL−1) and one was weakly resistant (EC50 = 10 µg mL−1). For the majority of these isolates EC50 values ranged from 0·06 to 4·22 µg mL−1 for boscalid and from 0·22 to 7·74 µg mL−1 for Pristine®. However, seven isolates resistant to pyraclostrobin were also highly resistant to boscalid and Pristine® and remained pathogenic on pistachio treated with Pristine®. Whereas strobilurin resistance is a common occurrence in Alternaria of pistachio, this is the first report of resistance to boscalid in field isolates of phytopathogenic fungi. No cross resistance between pyraclostrobin and boscalid was detected, suggesting that Pristine® resistance appears as a case of multiple resistance.

Using real-time PCR to survey frequency of azoxystrobin-resistant allele G143A in Alternaria populations from almond and pistachio orchards in California

Alternaria spp. cause leaf spot of almond and Alternaria late blight of pistachio in California, and azoxystrobin is a strobilurin fungicide that has been registered for the control of these diseases. To date, only a single point mutation of G143A in cytochrome b resulting to azoxystrobin resistance in Alternaria spp. was found in California. Based on this single point mutation, a real-time PCR assay was developed to quantify the frequency of the resistant allele G143A (FA) in pathogen samples taken from orchards. Forty-one almond and pistachio orchards were arbitrarily selected in eight counties of California. Fifty leaf lesions caused by Alternaria spp. per orchard were cut to extract the fungal DNA for a real-time PCR assay to determine the FA. About 88% of 41 surveyed orchards had Alternaria spp. with FA > 0.90, while six pistachio orchards showed a FA < 0.90. Therefore, azoxystrobin-resistant Alternaria populations are predominant in almond and pistachio orchards in California, and sprays of azoxystrobin to control Alternaria diseases are not recommended in these orchards. This study shows a potential use of a real-time PCR assay to efficiently quantify the frequency of azoxystrobin-resistant Alternaria spp. from large number of samples.

Characterization of iprodione-resistant Alternaria isolates from pistachio in California

Alternaria late blight caused by Alternaria spp. in the alternata, tenuissima, and arborescens species-groups is one of the most common fungal diseases of pistachio in California. In this study, a field iprodione-resistant (FIR) isolate of the arborescens species-group and a laboratory-induced iprodione-resistant (LIIR) isolate of the alternata species-group were characterized by fungicide and osmotic sensitivity, virulence on detached pistachio leaflets, and sequence of the coiled-coil region (six repeats of approximately 90-amino-acid domain) of the two-component histidine kinase ( HK) gene. Both FIR and LIIR isolates were sensitive to azoxystrobin and tebuconazole, and azoxystrobin-resistant isolates were sensitive to iprodione and tebuconazole. The LIIR isolate showed more sensitivity to osmotic stress than its wild-type parent. However, the FIR isolate did not show higher osmotic sensitivity compared to field iprodione-sensitive (FIS) isolates. Laboratory inoculation tests showed that both FIR and LIIR isolates remained highly virulent on pistachio. Analysis of DNA sequences of the HK coiled-coil region showed that there were no differences in deduced amino acid sequence of this region from the LIIR, FIR, and FIS Alternaria isolates from pistachio in California.

A real-time PCR assay for the detection of azoxystrobin-resistant Alternaria populations from pistachio orchards in California

Azoxystrobin-resistant populations of Alternaria spp. in the alternata, tenuissima, and arborescens species–groups, the causal agents of Alternaria late blight of pistachio, have been selected in pistachio orchards in California. The azoxystrobin resistance in Alternaria spp. was found to be correlative to a single point mutation resulting in the replacement of a glycine by an alanine at codon 143 (G143A) in the mitochondrial cytochrome b ( cyt b) gene. Based on this point mutation, a real-time PCR method was developed to rapidly detect azoxystrobin-resistant Alternaria populations in field leaf samples collected from pistachio orchards in California.

Resistance to azoxystrobin in Alternaria isolates from pistachio in California

Failure to control Alternaria late blight in a few California pistachio orchards was observed after only 3–4 years of consecutive applications of azoxystrobin-based fungicide programs. A total of 72 isolates of Alternaria alternata, Alternaria tenuissima, and Alternaria arborescens, the causal organisms of Alternaria late blight, were collected from pistachio orchards with (58 isolates) and without (14 isolates) a prior history of azoxystrobin applications. The sensitivity to azoxystrobin was determined in conidial germination assays. Isolates from orchards with a history of azoxystrobin applications had EC 50 values greater than 100 μg/ml, whereas isolates from orchards without a prior history of azoxystrobin usage had EC 50 values ranging from 0.008 to 0.045 μg/ml. Azoxystrobin resistance correlated with a single mutation in the cytochrome b (cyt b) gene causing a change of glycine to alanine at amino acid position 143. A pair of PCR primers AF and AR was developed that amplified a 226-bp DNA fragment of the cyt b gene containing the mutation site from all three Alternaria species but not from 30 other fungal species frequently found on pistachio. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis using the restriction enzyme Fnu4HI allowed differentiation of the PCR fragment of wild type cyt b gene from that of mutated gene. This method will aid in a fast detection of azoxystrobin resistance in these three Alternaria species.

Buried drip irrigation reduces fungal disease in pistachio orchards

Alternaria late blight, a fungal disease affecting both leaves and fruit, can lower the quality of pistachios and reduce grower profit. High humidity in orchards increases the magnitude and severity of the blight infections. One cause of high orchard humidity is the evaporation of water from the soil surface, which in turn is enhanced by irrigation systems that wet the surface. In this study, we tested the use of buried drip irrigation, which reduces orchard floor wetting, to see how well it controlled the disease. When compared with a traditional flood irrigation system, the buried drip system reduced orchard humidity and dew duration and increased temperature. This significantly reduced leaf symptoms of the disease and fruit infection at harvest. Additionally, more shells split open with the buried drip method, resulting in a higher yield of marketable pistachios.

Morphological, Pathogenic, and Molecular Characterization of Alternaria Isolates Associated with Alternaria Late Blight of Pistachio

ABSTRACT Alternaria isolates were obtained from various pistachio tissues collected in five orchards in California. For all isolates, morphological characteristics of the colony and sporulation apparatus were determined and compared with those of representative isolates of A. alternata, A. tenuissima, A. arborescens, and A. infectoria. A selection of the pistachio isolates and the representative Alternaria isolates were evaluated for pathogenicity to pistachio. Molecular characteristics of these isolates were determined using random amplified polymorphism DNA (RAPD) analysis, polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of nuclear intergenic spacer rDNA, and sequence analysis of nuclear internal transcribed spacer (ITS) rDNA. Based on morphological characteristics, the pistachio isolates were grouped as identical or very similar to either A. alternata, A. tenuissima, A. arborescens, or A. infectoria. Isolates from the alternata, tenuissima, and arborescens species-groups were pathogenic to pistachio and no significant differences in pathogenicity were observed. Isolates from the infectoria species-group were only weakly pathogenic to pistachio. Based on cluster analysis of RAPD and PCR-RFLP data, three distinct clusters were evident; the infectoria cluster, the arborescens cluster, and a combined alternata/tenuissima cluster. Based on analysis of ITS sequence data, the infectoria species-group was phylogenetically distinct from the other species-groups. Isolates of the alternata, tenuissima, and arborescens species-groups comprised a monophyletic clade in which the three species-groups could not be further resolved.

Plant Protection for Pistachio

Pistachio (Pistacia vera) was successfully introduced into California and initially touted as a tree nut crop with no disease or insect pests. Unfortunately, these expectations were dashed as a number of diseases and pests followed commercial plantings, making plant protection practices integral to production. Verticillium wilt (Verticillium dahliae) devastated early plantings but is now controlled with the use of resistant rootstocks. Botryosphaeria blight (Botryosphaeria dothidea) and alternaria late blight (Alternaria alternata) are recently arrived foliar fungal diseases that blight fruit clusters and defoliate trees, respectively, and multiple fungicide applications are needed for control. The conversion to low volume irrigation systems, specifically to drip or buried drip, has reduced disease. Pruning out botryosphaeria blight infections has reduced overwintering inoculum and disease, while current research aims at accurately predicting infection events to increase fungicide efficacy. A number of hemipteran insect pests have been associated with epicarp lesion: spring treatments have been replaced with dormant carbaryl and oil applications which are less toxic to beneficial insects while controlling phytocoris (Phytocoris californicus and P. relativus) and soft scale pests. Early season insect damage can be tolerated because trees compensate by maturing a higher percentage of remaining fruit kernels. Some mirid (Calocoris spp.) pests can be effectively reduced by eliminating alternate hosts in an effective weed control program. If lygus (Lygus hesperus) populations are present, weeds should not be disturbed from bloom until shell hardening to prevent movement by insects into the trees where feeding can result in epicarp lesion. Stink bugs (Pentatomidae) and leaffooted bugs (Leptoglossus clypealis and L. occidentalis) can penetrate the hardened shell and cause internal nut necrosis along with epicarp lesion. Trap crops are used to monitor pest populations in order to develop treatment thresholds. Degree-day based timing of treatments increase insecticide efficacy for the control of navel orangeworm (Amyelois transitella) and obliquebanded leafroller (Choristonuera rosaceana), but navel orangeworm populations are more effectively managed by destroying unharvested over wintering fruit. Bacillus thuriengiensis sprays, liquid-lime-sulfur, and biological control show promise in controlling obliquebanded leafroller.

Alternaria Late Blight (Alternaria alternata) Resistance in Pistachio (Pistacia vera) and Selection of Resistant Genotypes

Pistachio (Pistacia vera L.) progeny were evaluated at two locations in California for resistance to alternaria late blight caused by Alternaria alternata (Fries) Keissler in 1995 and 1997. Large differences in alternaria late blight infection among seedlings were observed. Narrow sense heritabilities based on half-sib analysis of 20 open pollinated families were 0.48 and 0.11 at Kearney Agricultural Center in 1995 and 1997, respectively, and 0.56 and 0.54 at the Wolfskill Experimental Orchard near Davis in 1995 and 1997, respectively. Differences among families to alternaria late blight infection were highly significant and associated with the female parents. Fifty-eight highly resistant seedlings were identified for future cultivar selection efforts.