Determining the Main Infection Courts in Sweet Cherry Trees of the Canker Pathogens Calosphaeria pulchella, Cytospora sorbicola, and Eutypa lata.

The major fungal canker pathogens causing branch dieback of sweet cherry trees in California include Calosphaeria pulchella, Cytospora sorbicola, and Eutypa lata. These pathogens have long been known to infect cherry trees mainly through pruning wounds. However, recent field observations revealed numerous shoots and fruiting spurs exhibiting dieback symptoms with no apparent pruning wounds or mechanical injuries. Accordingly, this study was conducted to assess the incidence of the three pathogens in symptomatic terminal shoots and dying fruiting spurs, in addition to the wood below pruning wounds in branches. Surveys were conducted in five sweet cherry orchards across three counties in California. We also investigated the possibility that leaf scars, bud scars, and wounds resulting from fruit picking could serve as infection courts for Cal. pulchella, Cyt. sorbicola, and E. lata by means of artificial inoculations in the field. Orchard surveys revealed that Cal. pulchella had the highest pathogen incidence below pruning wounds in branch samples, followed by Cyt. sorbicola and E. lata. Among terminal shoots with dieback symptoms and dying fruiting spurs, Cyt. sorbicola was the most prevalent, followed by Cal. pulchella. Results from field inoculations indicated that fruit-picking wounds could serve as important infection courts for Cal. pulchella, Cyt. sorbicola, and E. lata, with average pathogen recovery of 41.5, 63, and 36.2%, respectively. Results also indicated that leaf and bud scars could serve as an entry site for Cyt. sorbicola, although recovery was relatively low. The present study is the first to identify harvest-induced wounds on fruiting spurs of sweet cherry as an important infection court of Cal. pulchella, Cyt. sorbicola, and E. lata.

Identification and Pathogenicity of Fungal Species Associated with Branch Canker and Shoot Blight on Persimmons (Diospyros kaki) in California.

Persimmon is a relatively new crop to California agriculture. Asian persimmons (Diospyros kaki) are the dominant species commercially cultivated in the United States, primarily grown in California, covering approximately 1,153 ha of bearing trees. In the growing seasons of 2020 and 2021, unusual shoot blight and branch cankers were observed in several persimmon orchards in San Joaquin and Solano counties in California. The most prevalent symptoms were well-defined black discoloration in the cambium and streaking in the vascular tissues of green shoots. On woody branches and old pruning wounds, symptoms manifested as black wedge-shaped cankers. Isolations from affected tissues revealed the occurrence of Diaporthe species, including D. chamaeropis, D. foeniculina, and an undescribed Diaporthe sp. as the most frequent isolated pathogens, followed by Eutypella citricola and Phaeoacremonium iranianum. The isolates were identified through multilocus phylogenetic analyses using nucleotide sequences of the rDNA internal transcribed spacer, ß-tubulin, and translation elongation factor 1-alpha genes. To fulfill Koch's postulates, mycelium plugs of the various fungal species identified were inserted in 2-year-old branches of mature persimmon trees after making wounds using a corkborer in field conditions. Results showed that Diaporthe spp., E. citricola, and P. iranianum are the main causal agents of branch canker and shoot dieback of persimmon trees in California, with Diaporthe spp. being the most frequently isolated pathogen.

Field Evaluation of Fungicides for the Management of Neofabraea Leaf Lesion of Olive in California.

Field experiments were conducted during the fall-winter seasons of 2017 to 2018 and 2018 to 2019 to evaluate the efficacy of various fungicides to control Neofabraea leaf lesion of olive. Field trials were conducted in the highly susceptible cultivar Arbosana in a commercial, super-high-density orchard in San Joaquin County, California. Up to eight fungicidal products were applied using an air blast backpack sprayer, and their efficacy was compared with different application strategies. Results showed that most products were effective in reducing infection by the pathogens and limiting disease severity. Overall, best disease control was achieved by thiophanate-methyl, cyprodinil, difenoconazole + cyprodinil, and chlorothalonil, providing up to 75% reduction in disease severity. Copper hydroxide did not control the disease. In 2018 to 2019, the fungicides difenoconazole + cyprodinil and ziram were evaluated in additional field trials using different application strategies (single, dual, and combined applications) suitable for pathogen resistance management. Results showed that both products provided significant reduction in disease severity (∼50%), although no differences in efficacy were found between the two products nor between the different application strategies. Both products performed equally using one or two applications at 2-week intervals following harvest.

Floral Microbes Suppress Growth of Monilinia laxa with Minimal Effects on Honey Bee Feeding.

Management of Monilinia laxa, the causal agent of brown rot blossom blight in almond (Prunus dulcis), relies heavily on the use of chemical fungicides during bloom. However, chemical fungicides can have nontarget effects on beneficial arthropods, including pollinators, and select for resistance in the pathogen of concern. Almond yield is heavily reliant on successful pollination by healthy honey bees (Apis mellifera); thus, identifying sustainable, effective, and pollinator-friendly control methods for blossom blight during bloom is desirable. Flower-inhabiting microbes could provide a natural, sustainable form of biocontrol for M. laxa, while potentially minimizing costly nontarget effects on almond pollinators and the services they provide. As pollinators are sensitive to floral microbes and their associated taste and scent cues, assessing effects of prospective biocontrol species on pollinator attraction is also necessary. Here, our objective was to isolate and identify potential biocontrol microbes from an array of agricultural and natural flowering hosts and test their efficacy in suppressing M. laxa growth in culture. Out of an initial 287 bacterial and fungal isolates identified, 56 were screened using a dual culture plate assay. Most strains reduced M. laxa growth in vitro. Ten particularly effective candidate microbes were further screened for their effect on honey bee feeding. Of the 10, nine were found to both strongly suppress M. laxa growth in culture and not reduce honey bee feeding. These promising results suggest a number of strong candidates for augmentative microbial biocontrol of brown rot blossom blight in almond with potentially minimal effects on honey bee pollination.

Identification and Characterization of Phytophthora Species Associated with Crown and Root Rot of Pistachio Trees in California.

Pistachio is one of the most widely cultivated nut crops in California, with approximately 115,000 ha of bearing pistachio trees. In recent years, several orchards were identified, with declining trees leading to substantial tree losses. Symptoms included trees with poor vigor, yellowing and wilting of leaves, crown rot, and profuse gumming on the lower portion of trunks. Thirty-seven Phytophthora-like isolates were obtained from crown rot tissues in the rootstock of grafted pistachio trees and characterized by means of multilocus phylogeny comprising internal transcribed spacer rDNA, beta-tubulin, and mt cox1 sequence data. The analysis provided strong support for the delineation and identification of three Phytophthora species associated with declining pistachio trees, including P. niederhauserii, P. mediterranea, and Phytophthora taxon walnut. Pathogenicity studies in potted University of California Berkeley I (UCBI) rootstocks (clonal and seeded) confirmed that all three Phytophthora species can cause crown and root rot of pistachio, thus fulfilling Koch's postulates. The widespread occurrence of Phytophthora crown rot in recently planted pistachio orchards and the susceptibility of UCBI rootstocks suggest this disease constitute an emerging new threat to the pistachio industry of California. To the best of our knowledge, this study is the first to report P. niederhauserii, P. mediterranea, and Phytophthora taxon walnut as causal agents of crown and root rots of pistachio.

Evaluation of Pruning Wound Protection Products for the Management of Almond Canker Diseases in California.

Almond trunk and branch canker diseases constitute a major cause of tree mortality in California. Numerous fungal pathogens have been associated with these canker diseases and pruning wounds act as major infection courts. Before this study, there were no products registered in California for the management of these diseases. In this study, fungicidal products including synthetic chemistries, biocontrols, paint, and a sealant were evaluated for preventing fungal pathogen infection via pruning wounds. In four field trials conducted over two dormant seasons, 16 pruning wound treatments were tested using handheld spray applications against five almond canker pathogens, namely Botryosphaeria dothidea, Neofusicoccum parvum, Cytospora sorbicola, Ceratocystis destructans, and Eutypa lata. The fungicide thiophanate-methyl (Topsin M; United Phosphorus, Bandra West, Mumbai, India) provided 82% overall disease prevention against four fungal pathogens. The biological control agent, Trichoderma atroviride SC1 (Vintec; Bi-PA, Londerzeel, Belgium), tested at three application rates, resulted in 90 to 93% protection of pruning wounds in field trials, and for individual pathogens ranged from 81 to 100% protection for the three rates. At the time of this publication, Vintec is being considered for registration as a biological control product for the prevention of almond canker diseases, while Topsin M is recommended to growers for the prevention of almond canker diseases. This research indicates that effective protection of pruning wounds from infection by almond canker pathogens can be achieved with a one-time spray application of thiophanate-methyl or the biocontrol T. atroviride SC1 (recommended 2 g/liter) after pruning.

Pleurostoma Decline of Olive Trees Caused by Pleurostoma richardsiae in California.

A single fungal pathogen was consistently isolated from symptomatic wood of olive trees (Olea europaea) displaying branch and trunk cankers in superhigh-density orchards in the Sacramento and San Joaquin Valleys of California. Morphological characters of the pathogen included two distinct types of conidia (thick-walled, dark brown, and globose and thin-walled, hyaline, and oblong to ellipsoid) and three types of phialides, indicating a pleurostoma-like fungus. Phylogenetic results of four nuclear loci including the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) and partial sequences of the actin, beta-tubulin, and translation elongation factor 1-α genes confirmed the isolates as Pleurostoma richardsiae. Pathogenicity trials conducted in the field involving 2- to 3-year-old branches of three widely planted oil olive cultivars (Arbequina, Arbosana, and Koroneiki) satisfied Koch's postulates and confirmed the pathogenic nature of this species to cause the decline of olive trees in California. All three cultivars were equally susceptible to Pl. richardsiae, indicating no detectable resistance to the pathogen. Additional isolations from symptomatic hosts including almond, peach, pistachio, and plum, also confirmed this species, suggesting that Pl. richardsiae is widespread in agricultural systems and should be considered an emerging pathogen of fruit and nut crops in California.

Fungal Pathogens Associated With Canker Diseases of Almond in California.

Almond canker diseases are destructive and can reduce the yield as well as the lifespan of almond orchards. These diseases may affect the trunk and branches of both young and mature trees and can result in tree death soon after orchard establishment in severe cases. Between 2015 and 2018, 70 almond orchards were visited throughout the Central Valley of California upon requests from farm advisors for canker disease diagnosis. Two major canker diseases were identified, including Botryosphaeriaceae cankers and Ceratocystis canker. In addition, five less prevalent canker diseases were identified, including Cytospora, Eutypa, Diaporthe, Collophorina, and Pallidophorina canker. Seventy-four fungal isolates were selected for multilocus phylogenetic analyses of internal transcribed spacer region ITS1-5.8S-ITS2 and part of the translation elongation factor 1-α, ß-tubulin, and glyceraldehyde 3-phosphate dehydrogenase gene sequences; 27 species were identified, including 12 Botryosphaeriaceae species, Ceratocystis destructans, five Cytospora species, Collophorina hispanica, four Diaporthe species, two Diatrype species, Eutypa lata, and Pallidophorina paarla. The most frequently isolated species were Ceratocystis destructans, Neoscytalidium dimidiatum, and Cytospora californica. Pathogenicity experiments on almond cultivar Nonpareil revealed that Neofusicoccum parvum, Neofusicoccum arbuti, and Neofusicoccum mediterraneum were the most virulent. Botryosphaeriaceae cankers were predominantly found in young orchards and symptoms were most prevalent on the trunks of trees. Ceratocystis canker was most commonly found in mature orchards and associated with symptoms found on trunks or large scaffold branches. This study provides a thorough examination of the diversity and pathogenicity of fungal pathogens associated with branch and trunk cankers of almond in California.

Macrophomina Crown and Root Rot of Pistachio in California.

In this study, declining pistachio rootstocks were detected in newly planted commercial pistachio orchards in Kern County, California. Symptoms were characterized by wilted foliage combined with crown rot in the rootstock. From diseased trees, 42 isolates were obtained, and all had similar cultural and morphological characteristics of Macrophomina phaseolina. Analyses of nucleotide sequences of three gene fragments, the internal transcribed spacer region (ITS1-5.8S-ITS2), partial sequences of ß-tubulin, and translation elongation factor 1-α (TEF1) confirmed this identification, and 20 representative isolates are presented in the phylogenetic study. Testing of Koch's postulates showed that M. phaseolina, when inoculated to stems and roots of the pistachio rootstocks using mycelial plugs or a microsclerotial suspension, is indeed pathogenic to this host. The widely used clonal University of California Berkeley I (UCBI) rootstock appeared highly susceptible to M. phaseolina, suggesting that this pathogen is an emerging threat to the production of pistachio in California. This study confirmed the association of M. phaseolina with the decline of pistachio trees and represents the first description of this fungus as a crown rot-causing agent of pistachio in California.

Identification and Characterization of Neofabraea kienholzii and Phlyctema vagabunda Causing Leaf and Shoot Lesions of Olive in California.

California produces over 95% of the olives grown in the United States. In 2017, California's total bearing acreage for olives was 14,570 hectares producing 192,000 tons of olives valued at $186.6 million. During the early spring of 2016, unusual leaf and shoot lesions were detected in olive trees from superhigh-density orchards in the Northern San Joaquin and Sacramento valleys of California. Affected trees displayed numerous leaf and shoot lesions developing at wounds created by mechanical harvesters. The 'Arbosana' cultivar was highly affected by the disease, whereas the disease was sporadic in 'Arbequina' and not found in 'Koroneiki' cultivar. Two fungal species, Neofabraea kienholzii and Phlyctema vagabunda, were found to be consistently associated with the disease, and Koch's postulates were completed. Species identity was confirmed by morphology and molecular data of the partial large subunit rDNA, the internal transcribed spacer region, and partial beta-tubulin region. The disease signs and symptoms are described and illustrated.

Identification and Pathogenicity of Fungal Species Associated with Canker Diseases of Pistachio in California.

A survey was conducted during 2015 and 2016 in pistachio orchards throughout the San Joaquin Valley of California to investigate the occurrence of canker diseases and identify the pathogens involved. Cankers and dieback symptoms were observed mainly in orchards aged >15 years. Symptoms of canker diseases included brown to dark brown discoloration of vascular tissues, wood necrosis, and branch dieback. In total, 58 fungal isolates were obtained from cankers and identified based on multilocus phylogenetic analyses (internal transcribed spacer, glyceraldehyde 3-phosphate dehydrogenase, ß-tubulin, calmodulin, actin 1, and translation elongation factor 1α) representing 11 fungal species: Colletotrichum karstii, Cytospora californica, Cytospora joaquinensis, Cytospora parapistaciae, Cytospora pistaciae, Diaporthe ambigua, Didymella glomerata, Diplodia mutila, Neofusicoccum mediterraneum, Phaeoacremonium canadense, and Schizophyllum commune. Pathogenicity tests conducted in the main pistachio cultivars Kerman, Golden Hills, and Lost Hills using the mycelium-plug method indicated that all fungal species were pathogenic to Pistacia vera. All species tested caused cankers in pistachio branches, although virulence among species varied from high to moderate. Overall, N. mediterraneum and Cytospora spp. were the most widespread and virulent species associated with canker diseases of pistachio in California.

Characterization of Fusarium and Neocosmospora Species Associated With Crown Rot and Stem Canker of Pistachio Rootstocks in California.

California produces 99.1% of pistachios grown in the United States, and diseases affecting pistachio rootstocks represent a constant challenge to the industry. Field surveys of fungi associated with pistachio rootstocks with symptoms of crown rot and stem canker in three central California counties followed by phylogenetic analyses of translation elongation factor 1-α and second largest subunit of RNA polymerase II gene fragments identified three Fusarium species (Fusarium equiseti, Fusarium oxysporum, and Fusarium proliferatum) and two Neocosmospora species (Neocosmospora falciformis and Neocosmospora solani). F. oxysporum and N. falciformis were the fungal species most frequently recovered from symptomatic pistachio trees. Inoculations of detached twigs of cultivar Kerman pistachio Pioneer Gold I and clonal University of California, Berkeley I (UCBI) rootstocks showed that all five species could colonize pistachio wood and cause vascular discolorations. Pathogenicity tests in potted pistachio trees completed Koch's postulates and confirmed that F. oxysporum, F. proliferatum, N. falciformis, and N. solani were capable of producing rot and discoloration in stems of clonal UCBI rootstocks, the most widely planted pistachio rootstock in California. To our knowledge, this study is the first to present insights into the biodiversity and biology of Fusarium and Neocosmospora species associated with pistachio trees in California.

Identification, Pathogenicity, and Spore Trapping of Colletotrichum karstii Associated with Twig and Shoot Dieback in California.

Colletotrichum Corda, 1831 species are well-documented pathogens of citrus that are associated with leaf and fruit anthracnose diseases. However, their role in twig and shoot dieback diseases of citrus has recently become more prominent. Recent surveys of orchards in the Central Valley of California have revealed C. gloeosporioides and a previously undocumented species, C. karstii, to be associated with twig and shoot dieback. Pathogenicity tests using clementine (cv. 4B) indicated that both C. karstii and C. gloeosporioides are capable of producing lesions following inoculation of citrus stems. Pathogenicity tests also revealed C. karstii to be the most aggressive fungal species producing the longest lesions after 15 months. The majority of spores trapped during this study were trapped during or closely following a precipitation event with the majority of spores being trapped from January through May. These findings confirm C. karstii as a new pathogen of citrus in California.

Molecular phylogeny of Cytospora species associated with canker diseases of fruit and nut crops in California, with the descriptions of ten new species and one new combination.

Cytospora species are destructive canker and dieback pathogens of woody hosts in natural and agroecosystems around the world. In this genus, molecular identification has been limited due to the paucity of multi-locus sequence typing studies and the lack of sequence data from type specimens in public repositories, stalling robust phylogenetic reconstructions. In most cases a morphological species concept could not be applied due to the plasticity of characters and significant overlap of morphological features such as spore dimensions and fruiting body characters. In this study, we employed a molecular phylogenetic framework with the inclusion of four nuclear loci (ITS, translation elongation factor 1-alpha, actin, and beta-tubulin) to unveil the biodiversity and taxonomy of this understudied important genus of plant pathogens. Phylogenetic inferences based on 150 Californian isolates revealed 15 Cytospora species associated with branch and twig cankers and dieback of almond, apricot, cherry, cottonwood, olive, peach, pistachio, plum, pomegranate, and walnut trees in California. Of the 15 species recovered in this study, 10 are newly described and typified, in addition to one new combination. The pathogenic status of the newly described Cytospora species requires further investigation as most species were associated with severe dieback and decline of diverse and economically important fruit and nut crops in California.

Neoscytalidium dimidiatum Causing Canker, Shoot Blight and Fruit Rot of Almond in California.

Almond trees with trunk and branch cankers were observed in several orchards across almond-producing counties in California. Symptoms of cankers included bark lesions, discoloration of xylem tissues, longitudinal wood necrosis, and extensive gumming. Spur and shoot blight associated with rotted fruit were detected in two orchards in Kern County. The fungus Neoscytalidium dimidiatum was consistently recovered from the various cankers, infected fruit, and blighted shoots and its identity was confirmed based on phylogenetic and morphological studies. Phylogenetic analyses of the internal transcribed spacer, translation elongation factor 1-α, and ß-tubulin genes comparing 47 strains from California with reference specimens within the family Botryosphaeriaceae and coupled with detailed morphological observations validated the identity of the pathogenic fungus. Pathogenicity tests conducted in the field using 1- to 2-year-old branches inoculated with mycelium plugs or conidial suspensions and attached fruit inoculated with conidial suspensions fulfilled Koch's postulates. N. dimidiatum appeared highly virulent in almond-producing cankers of up to 22 cm in length within 4 weeks using mycelium plug inoculations as well as severe fruit rot combined with spur blight on the fruit-bearing spurs. This study reports, for the first time, the fungus N. dimidiatum as a pathogen of almond in California causing canker, shoot blight, and fruit rot. Disease symptoms are described and illustrated.

Novel TULP1 mutation causing leber congenital amaurosis or early onset retinal degeneration.

PURPOSE: To report a large, consanguineous Algerian family affected with Leber congenital amaurosis (LCA) or early-onset retinal degeneration (EORD). METHODS: All accessible family members underwent a complete ophthalmic examination, and blood was obtained for DNA extraction. Homozygosity mapping was performed with markers flanking 12 loci associated with LCA. The 15 exons of TULP1 were sequenced. RESULTS: Seven of 30 examined family members were affected, including five with EORD and two with LCA. All patients had nystagmus, hemeralopia, mild myopia, and low visual acuity without photophobia. Fundus features were variable among EORD patients: typical spicular retinitis pigmentosa or clumped pigmented retinopathy with age-dependent macular involvement. A salt-and-pepper retinopathy with midperipheral retinal pigment epithelium (RPE) atrophy was present in the older patients with LCA, whereas the retina appeared virtually normal in the younger ones. Both scotopic and photopic electroretinograms were nondetectable. Fundus imaging revealed a perifoveal ring of increased fundus autofluorescence (FAF) in the proband, and optical coherence tomography disclosed a thinned retina, mainly due to photoreceptor loss. Linkage analysis identified a region of homozygosity on chromosome 6, region p21.3, and mutation screening revealed a novel 6-base in-frame duplication, in the TULP1 gene. CONCLUSIONS: Mutation in the TULP1 gene is a rare cause of LCA/EORD, with only 14 mutations reported so far. The observed intrafamilial phenotypic variability could be attributed to disease progression or possibly modifier alleles. This study provides the first description of FAF and quantitative reflectivity profiles in TULP1-related retinopathy.