First Report of Geotrichum candidum Causing Rubbery Rot on Potato in Arizona.

In January 2023, stand loss due to seed decay (<3% incidence) was reported in a commercial seed potato field in Cochise County, Arizona. Several rotted 'Ciklamen' tubers with wilting shoots were submitted to the University of Arizona Extension Plant Pathology Laboratory for diagnosis. External symptoms included sunken black-colored lesions on affected tubers; white fungal-like growth was also present on the surface of the more severely affected tubers. Internal tissues were watery, rubbery-textured, and spongy with slight gray discoloration. Isolation from the necrotic tubers on PDA yielded yeast-like fungal colonies. Two derived fungal isolates were subcultured on PDA at room temperature for three days, and colonies had a white, dense, and creamy appearance. Hyphae were hyaline, septate, dichotomously branched, and broken into chains of cylindrical to oval-shaped, single-celled arthrospores ranging in size from 3.8-5.3 µm × 5.4-12.5 µm (n = 20). These morphological characteristics matched those of Geotrichum candidum (Carmichael, 1957). Other common potato pathogens, including Pythium, Phytophthora, and Fusarium, were not detected in the samples. For molecular identification, genomic DNA was extracted from the mycelia of two isolates using the DNeasy Plant Pro Kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. Partial DNA sequences of the internal transcribed spacer (ITS) region of rDNA and elongation factor 1-alpha (EF-1α) gene were amplified and sequenced using primers ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-1567R (Carbone and Kohn 1999) and EF1-1018/EF1-1620R (Stielow et al., 2015). The resulting sequences showed no difference between the isolates and were deposited under the GenBank Accession Numbers OQ690039 and OQ981192. Based on BLASTn analysis, ITS sequence OQ690039 (369-bp) had a 99.46% match with Geotrichum candidum accessions MN861070 and KY103456. EF-1α sequence OQ981192 (843-bp) showed 100% identity with G. candidum accessions MT346368 of the G. candidum isolates responsible for sour rot of strawberries in Florida (Alonzo et al., 2020). A pathogenicity test was conducted twice on ten healthy 'Ciklamen' tubers, surface sterilized using 10% sodium hypochlorite solution, and rinsed copiously in sterilized water. A conidial suspension (106 conidia/ml) of the isolated fungus was sprayed onto the surface of the tubers. The control consisted of ten tubers that were mock-sprayed with sterilized water. All tubers were incubated in separate sterilized plastic bags in the dark at room temperature for four weeks and examined daily for symptom development. Symptoms of rubbery texture and fluid leaking when cut, and a distinctive smell, were produced on the inoculated tubers. In contrast, the controls did not show any symptoms. These symptoms were similar to those observed on naturally infected tubers. The fungus was re-isolated from symptomatic tissues of inoculated tubers and identified by PCR as G. candidum, thus fulfilling Koch's postulates. To our knowledge, this is the first report of G. candidum as a potato pathogen in Arizona. Rubbery rot of potatoes by G. candidum has been reported previously in Idaho (Duellman et al., 2020) and Michigan (Willbur et al., 2021). Arizona annually grows approximately 1,000 hectares of seed potatoes shipped to various states. Therefore, growers must obtain a correct diagnosis to avoid spreading the disease despite the low incidence of disease.

First Report of Neodeightonia phoenicum Causing Black Scorch and Rot Disease on Date Palms in Arizona.

Phoenix dactylifera L. is an economically and aesthetically important tree in the southwestern US. Approximately 4900 ha of dates are commercially grown for its edible fruit in the US, including about 1600 ha in the Yuma area and the Hyder Valley of Arizona (USDA, 2023). In October 2022, a severe rot was observed on three date palms in the Phoenix Metropolitan area. Early symptoms were brown spots that turned to a black scorch appearance extending along the leaf base and rachis, leading to the lower fronds' wilting, drying, and folding. As the disease progressed upwards, the terminal bud became necrotic and eventually collapsed. Isolation from the necrotic leaf lesions on a potato dextrose agar (PDA) consistently yielded a fast-growing fungus that was initially white with abundant fluffy aerial mycelium, which gradually turned dark olivaceous after growing at 22-25oC under 12 h light for a week. Pycnidial conidiomata formed on pine needles in a water agar were black and globose. Conidiogenous cells were hyaline and cylindrical. The conidia exhibited a thick-walled, ovoid to ellipsoid morphology, initially appearing hyaline and aseptate and transitioned to 1-septate with a dark brown, striated appearance, measuring 19.6 to 23.0 µm x 10.3 to 12.2 µm (n = 20). For molecular identification, genomic DNA was extracted from the mycelia of two isolates. Partial DNA sequences of the internal transcribed spacer (ITS) region of rDNA and ß-tubulin (TUB) gene were amplified and sequenced using primers ITS5/ITS4 (White et al. 1990) and Bt2a/Bt2b (Glass and Donaldson 1995). The resulting sequences of ITS (PP346666) and TUB (PP372690) were deposited in the GenBank. A BLASTn search of ITS and TUB sequences revealed a 99 to 100% similarity with the sequences (JX456475, KF766198, and OK338070) of Neodeightonia phoenicum strains causing palm rot in Greece (Ligoxigakis et al. 2013), leaf spot on pygmy date palm in China (Zhang and Song 2022), and an ex-type CBS 122528 culture. Based on these morphological and molecular data, the fungus was identified as N. phoenicum. A pathogenicity test was conducted twice in a greenhouse (daily temperatures:18 ~ 30 oC, relative humidity: 45% ~ 95%) on 4 healthy 1-year-old date palm plants. The petioles of 3 older leaves per plant were wounded by pricking the epidermis of the leaf with a needle (ca 20 pricks per petiole) and inoculated with agar discs from a 4-day-old PDA culture of the fungus. The control consisted of 4 mock-inoculated plants by placing plain PDA on the wounds of leaf petioles. Five weeks after inoculation, all the inoculated leaves showed symptoms of black scorch, petiole rot, and leaf necrosis, which were the same as those symptoms observed on the original diseased trees, while the controls did not show any symptoms. The fungus was re-isolated and confirmed as N. phoenicum by morphology. N. phoenicum has been reported to cause leaf spot, shoots blights, stalk and root rots as well as black scorch on different palm species all over the world. However, to our knowledge, this is the first report of N. phoenicum causing black scorch and rot disease in Arizona. The possible spread of N. phoenicum could have a significant economic impact and requires immediate attention through suitable disease management initiatives.

Phytophthora capsici, 100 Years Later: Research Mile Markers from 1922 to 2022.

In 1922, Phytophthora capsici was described by Leon Hatching Leonian as a new pathogen infecting pepper (Capsicum annuum), with disease symptoms of root rot, stem and fruit blight, seed rot, and plant wilting and death. Extensive research has been conducted on P. capsici over the last 100 years. This review succinctly describes the salient mile markers of research on P. capsici with current perspectives on the pathogen's distribution, economic importance, epidemiology, genetics and genomics, fungicide resistance, host susceptibility, pathogenicity mechanisms, and management.

First Report of Cladosporium ramotenellum Causing Sooty Spot on Postharvest Clementines in the United States.

In 2022, post-harvest symptoms of black spots were observed at an incidence of 2-5% on easy peeling clementines (citrus × clementina) in a fresh fruit market in Phoenix metropolitan area, Arizona. Black lesions on the fruit rind were superficial, circular, dry, and firm with gray sporulation. Gray or black aerial mycelium was also noticed on top of the black spots. Black spots were distributed over the entire fruit surface without any regular patterns. Maceration of rind and flesh was also noticed under refrigerated storage conditions. Two isolates were obtained (S13 and S14) and cultured on potato dextrose agar (PDA) at 25oC for 14 days. The colony color and texture of both isolates were identical on PDA: olivaceous black with abundant sporulation, margin entire edge to slightly undulate. Conidia were 0-1 septate and globose or ellipsoid with sizes ranging from 4-10 × 2-4 µm (n = 15). Ramoconida were also 0-1 septate and ellipsoid to cylindrical with sizes ranging from 15-30 × 2-5 µm (n = 15). Conidiophore had a width of 3-6 µm (n = 15). Isolates were identified as Cladosporium ramotenellum based on these morphological features (Bensch et al. 2012). The IDs of these two isolates were further confirmed by genomic DNA isolation, PCR amplification and sequencing of the ITS1-5S-ITS2-28S region of rDNA (V9G/LR5 de Hoog and Gerrits van den 1998, primers ITS4/ITS5 White et al.), actin gene (ACT-512F/ACT-783R, Carbone and Kohn 1999), and elongation factor 1α gene (EF1-1018F/EF1-1620R, Stielow 2015). Sequences of the two isolates were identical and thus only one sequence of each gene was deposited in the GenBank. A BLASTn search of actin sequence (233-bp, OQ185511) revealed 99.1% match with ex-type sequence EF 679538 (strain CBS:121628) of C. ramotenellum holotype (query coverage: 97%). BLASTn analysis of a portion of EF-1α gene (579-bp, OQ185512) revealed more than 99.7% similarity with sequences KU933429 (ATCC strain 16022) and MT881827 (strain 18EPLE003) of C. ramotenellum (query coverage: 100%). The ITS sequence (1519-bp, OQ236707) was identical to the ITS sequences of C. ramotenellum strains in easy peeler mandarins from Peru (Murciano et al. 2021). Pathogenicity tests were carried out twice on fresh easy-peeling clementine fruit. The inocula (1 x 105 spores/ml) were prepared in sterile distilled water containing 0.1% Tween 20 (TW) by mixing the conidial suspensions of two isolates from 7-day-old PDA cultures. Ten fruit were washed, surface sanitized with 70% ethanol, and wound-inoculated by immersing five fruit in spore suspension for 1 min. Five control fruit were wound-inoculated with TW. Inoculated and control fruit were stored in separate zip-lock bags for one week. Black spots resembling those observed on naturally infected fruit were present on inoculated fruit, while control fruit remained symptomless. C. ramotenellum was reisolated and was morphologically identical to the original isolates, thus completing Koch's postulates. C. ramotenellum has been reported as a fungal pathogen causing sooty spots on easy peeler mandarins from Peru (Murciano et al. 2021). To our knowledge, this is the first report of C. ramotenellum causing postharvest sooty spot and decay on clementines in the United States. Results show that infected fruit is potentially a pathogen source for long-distance dispersal. This occurrence was communicated to the state regulatory agencies for regulatory actions on imports of citrus fruit from countries with the occurrence of C. ramotenellum.

First Report of Globisporangium heterothallicum Causing Seedling Disease on Guayule in Arizona.

Guayule (Parthenium argentatum A. Gray) is known for producing low-allergenic latex that is used in high end rubber products for medical use such as rubber gloves, catheters, and condoms. Currently, there are growing efforts from tire industry to commercialize guayule for rubber production in Arizona. During May 2019, wilting and death of c. 25% of seedling plants were observed in direct-seeded guayule fields in central Arizona. Symptoms of root rots and hypocotyl constriction were observed on affected seedling plants. To identify the causal agent, four symptomatic plants were collected to isolate the putative pathogen. Small pieces of symptomatic root (2-5 mm) were surface sterilized in 0.6% sodium hypochlorite for 1 min, rinsed copiously in sterile distilled water, blotted dry, and plated on 10% clarified V8-PARP (Jeffers and Martin 1986). Four oomycete-like isolates with abundant hyphal swellings were purified by transferring tips of single hypha onto new 20% CV8 plates and incubating at 23°C for one week. Sporangia were formed abundantly, globose or lemon-shaped (average 20 ± 4 × 20 ± 4 µm, n = 15). Isolates did not produce oospores (heterothallic). Genomic DNA was extracted from the mycelia of two isolates using DNeasy Plant Pro Kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. The internal transcribed spacer (ITS) region of rDNA and mitochondrially encoded cytochrome c oxidase 1 (cox 1) gene were amplified with primers ITS1/ITS4 (White et al., 1990) and OomCoxI-Levup/OomCoxI-Levlo (Martin and Tooley, 2003; Robideau et al., 2011) and the resulting amplicons were sequenced (GenBank Accession No. OL514636 and OL539842). A BLASTn search of 808-bp amplicon (OL514636) revealed 100% match with ITS sequences MT039880 which was G. heterothallicum causing root and crown rot of pepper in Turkey. BLAST analysis of the 658-bp amplicon (OL539842) showed 99.39 % identity with the COX 1 sequence of G. heterothallicum from tomato in Australia (MT981128). To fulfill Koch's postulates, pathogenicity tests were conducted twice on 2-week-old 'Az 2' guayule plants grown in 1.9-liter pots filled with a steam-disinfested potting mix. Pots were placed in a plastic container and watered three times a week by flooding, to create waterlogged conditions. Plants were maintained in a greenhouse and fertilized weekly with a 20-20-20 fertilizer at 1mg/ml. Twenty plants in 5 pots (4 plants/pot) were challenged with a G. heterothallicum isolate by drenching pot with 50 ml of a 1×106 zoospore/ml suspension. Twenty plants in 5 pots, serving as a control, received each 50 ml of distilled water. Symptoms of wilting and water-soaked root rot, and plant death were observed 2 weeks afterward, whereas control plants remained asymptomatic. G. heterothallicum was reisolated from necrotic roots of inoculated plants but not from control plants. G. heterothallicum has been increasingly reported as a pathogen of damping-off or root and crown rot on hosts such as alfalfa in Minnesota (Berg et al., 2017), soybean in Pennsylvania (Coffua et al., 2016), spinach in Sweden (Larsson, 1994), corn in China (Gan, et al., 2010), pepper in Turkey (Dervis, et al., 2020). To our knowledge, this is the first report of G. heterothallicum causing guayule seedling diseases in the United States. The presence of broad-host-range pathogen G. heterothallicum suggests that new strategies are needed for managing this pathogen to increase stands in direct-seeded guayule production system.

Meta-Analysis of the Field Efficacy of Seed- and Soil-Applied Nematicides on Meloidogyne incognita and Rotylenchulus reniformis Across the U.S. Cotton Belt.

Meta-analysis was used to compare yield protection and nematode suppression provided by two seed-applied and two soil-applied nematicides against Meloidogyne incognita and Rotylenchulus reniformis on cotton across 3 years and several trial locations in the U.S. Cotton Belt. Nematicides consisted of thiodicarb- and fluopyram-treated seed, aldicarb and fluopyram applied in furrow, and combinations of the seed treatments and soil-applied fluopyram. The nematicides had no effect on nematode reproduction or root infection but had a significant impact on seed cotton yield response ([Formula: see text]), with an average increase of 176 and 197 kg/ha relative to the nontreated control in M. incognita and R. reniformis infested fields, respectively. However, because of significant variation in yield protection and nematode suppression by nematicides, five or six moderator variables (cultivar resistance [M. incognita only], nematode infestation level, nematicide treatment, application method, trial location, and growing season) were used depending on nematode species. In M. incognita-infested fields, greater yield protection was observed with nematicides applied in furrow and with seed-applied + in-furrow than with solo seed-applied nematicide applications. Most notable of these in-furrow nematicides were aldicarb and fluopyram (>131 g/ha) with or without a seed-applied nematicide compared with thiodicarb. In R. reniformis-infested fields, moderator variables provided no further explanation of the variation in yield response produced by nematicides. Furthermore, moderator variables provided little explanation of the variation in nematode suppression by nematicides in M. incognita- and R. reniformis-infested fields. The limited explanation by the moderator variables on the field efficacy of nematicides in M. incognita- and R. reniformis-infested fields demonstrates the difficulty of managing these pathogens with nonfumigant nematicides across the U.S. Cotton Belt.

First Report of Crown and Root Rot Caused by Pythium myriotylum on Hemp (Cannabis sativa) in Arizona.

During August and September 2020, symptoms of leaf chlorosis, stunting, and wilting were observed on indoor hemp plants (Cannabis sativa L. cv. 'Wedding Cake') in a commercial indoor facility located in Coolidge, Arizona. Plants were grown in soilless coconut coir growing medium (Worm Factory COIR250G10), watered with 1.5 to 2.1 liters every 24 h through drip irrigation, and supplemented with 18 h of lighting. About 35% of plants displayed symptoms as described above and many symptomatic plants collapsed. To identify the causal agent, crown and root tissues from four symptomatic plants were harvested and rinsed with tap water. Tissue fragments (approx. 2 to 4 mm in size) were excised from the margins of the stem and root lesions, surface sterilized in 0.6% sodium hypochlorite for 1 min, rinsed well in sterile distilled water, blotted dry, and plated on potato dextrose agar (PDA) and on oomycete-selective clarified V8 media containing pimaricin, ampicillin, rifampicin, and pentachloronitrobenzene (PARP). Plates were incubated at room temperature (21-24 oC). Five isolates resembling Pythium were transferred after 3 days and maintained on clarified V8 media. Morphological characteristics were observed on grass blade cultures (Waterhouse 1967). Grass blades were placed on CV8 inoculated with the isolate. After a 1-day incubation at 25°C, the colonized blades were transferred to 8 ml of soil water extract in a Petri dish. Ten sporangia and oogonia were selected randomly and their diameters were measured under the microscope. Sporangia were mostly filamentous, undifferentiated or inflated lobulate, ranging from 7 to 17 µm in diameter. Knob-like appressoria were observed on branching clusters. Bulbous-like antheridia were formed on branched stalk with 1-8 antheridia per oogonium. Globose oogonia were terminal or intercalary and ranged from 21 to 33 µm in diameter. Globose oospores were mostly aplerotic and ranged from 15 to 21 µm in diameter. Based on these morphological characteristics, isolates were tentatively identified as Pythium myriotylum (Watanabe, 2002). Genomic DNA was extracted from mycelial mats of two isolates using DNeasy Plant Pro Kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. The internal transcribed spacer (ITS) region of rDNA was amplified with primers ITS1/ITS4 and two identical nucleotide sequences were obtained and deposited under accession number MW380925. A BLASTn search revealed ≥ 98% query coverage and 100% match with sequences HQ237488.1, KY019264.1, and KM434129, which were isolates of P. myriotylum from palm, tobacco, and ginger, respectively. To fulfill Koch's postulates, pathogenicity tests were conducted with 2 isolates using plants of 'Wedding Cake' grown in 12 1.9-liter pots filled with a steam-disinfested potting mix (Sungro Professional Growing Mix). Pots were placed in a plastic container and watered to flooding three times a week. Plants were maintained in a greenhouse with 18 h/10 h day/night supplemental light cycle (15-28 oC). Plants were fertilized weekly with Peters Professional fertilizer at 1mg/ml. Four plants were inoculated with each isolate at three weeks after seed sowing by placing two 5-mm mycelial plugs from active growing 4 days-old cultures on PDA media adjacent to the main root mass at an approximately 3 cm depth. Four plants were inoculated with blank PDA plugs as controls. Symptoms of leaf chlorosis, crown rot and wilting were observed after four weeks while control plants remained symptomless. P. myriotylum was re-isolated from necrotic roots of inoculated plants after surface-sterilization, but not from control plants. The pathogenicity test was repeated once. While P. myriotylum often occurs in warmer regions and has a wide host range of >100 host plant species including numerous economically important crops (Wang et al., 2003), there are only two reports of this pathogen on indoor hemp plants in a greenhouse in Connecticut (McGehee et al., 2019) and in Canada (Punja et al., 2019). This is the first report of P. myriotylum causing root and crown rot of indoor hemp in Arizona. A more careful water management in soilless growth medium to reduce periods of saturation would minimize the risk of Pythium root rot in indoor hemp production.

First Report of 'Candidatus Phytoplasma trifolii' Related Strain Associated with Yellowing and Witches'-Broom of Industrial Hemp (Cannabis sativa) in Arizona.

In Arizona, industrial hemp (Cannabis sativa) is a newly cultivated crop for fiber, oil, cosmetic products, and health food. During July to September 2020, two fields of industrial hemp crops were identified in southern Arizona with 10 to 30% incidence of plants showing witches' broom. Disease incidence was assessed by counting symptomatic plants in 4 randomly selected rows of 25 plants in each field. Symptoms ranged from leaf mottling and yellowing on mildly affected plants to leaf curling and shortened internode length of stem on severely affected plants (Fig. 1). Shoots were randomly collected from eight symptomatic plants and three asymptomatic plants in the same area. Genomic DNA was extracted from 200 mg of each sample using DNeasy Plant Pro Kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. Phytoplasma was tested by a real-time PCR assay and TaqMan probe targeting the 23S ribosomal RNA gene that detects a wide range of known Phytoplasmas (Hodgetts et al., 2009). Beet curly top virus (BCTV) was targeted using BCTV-specific primers BCTV1 and BCTV2 following a method by Rondon (Rondon et al., 2016). BCTV was not detected in the plants, but Phytoplasmas were detected in all eight symptomatic plants, but not in the three control plants. The positive DNA samples were used to identify the phytoplasma by nested PCR using universal phytoplasma-specific primer pairs P1/P6 (Deng, S. et al. 1991) and R16F2n/R16R2 (Gundersen et al., 1996) targeting the 16S rRNA gene and the resulting 1.25 kb fragment in 4 positive samples was subjected to Sanger sequencing (Eton Bioscience, San Diego). All 4 sequences were identical and deposited in GenBank under accession MW981356. BLASTn results indicated 100% identity with that of several 'Candidatus Phytoplasma trifolii' strains on potato (KR072666, KF178706) in Washington and chile peppers (HQ436488) in New Mexico. It also shared 99.84% identity with the sequence of the reference strain of Candidatus Phytoplasma trifolii' (AY390261) that caused clover proliferation. The phytoplasma AZH1 was classified as a member of subgroup A within group16SrVI using iPhyClassifier, an interactive online tool for phytoplasma classification and taxonomic assignment (Zhao et al., 2013). Phylogenetic analysis revealed that the phytoplasma AZH1 clustered with other isolates of 'Candidatus Phytoplasma trifolii' (Fig. 2), including the strain NV1 associated with witches' broom on C. sativa in Nevada (Feng et al. 2019). This is the first report of 'Candidatus Phytoplasma trifolii' related strain associated with yellowing and witches' broom on hemp in Arizona. This finding is significant as the observation of symptoms at 30% incidence in one field suggested that the identified pathogen may pose a significant threat to the production of industrial hemp production in Arizona.

First Report of Fomitopsis meliae Causing Brown Wood Rot on Living Lemon Trees in Arizona and California.

Arizona is one of the largest producers of premium lemons for fresh market in the world. In 2018, 54,000 metric tons of lemons were produced on more than 7,300 acres. In recent years, wood rot diseases have become increasingly important in desert citrus production, with an incidence rate of 70 to 100% in some lemon orchards in Yuma, and lower rates in lemon orchards in the California desert. In 2018 and 2019, A brown wood rot showing symptoms of leaf chlorosis, branch gummosis and wilt, and branch breakage was the most common disease in many lemon orchards. Where disease was observed, a white mycelial mass often covered split exposed internal wood and basidiocarps were found on fallen decaying wood on orchard floors. The fungal colony, consistently isolated from decaying wood on malt extract agar or potato dextrose agar (PDA), was white, dense, and cottony-floccose. The 7-day-old culture had a distinct mushroom odor and hyphae were hyaline, thin-walled, and clamped generative. Genomic DNA was extracted from the mycelia of three isolates using DNeasy Plant Pro Kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. The internal transcribed spacer (ITS) region of rDNA was amplified with primers ITS1/ITS4 (White et al., 1990). The resulting three nucleotide sequences were identical and deposited under accession number MW221272 in GenBank. A BLASTn search revealed 97.45% and 99.94% match with sequences HQ248221.1 and KT718002.1, respectively, which were isolates of Fomitopsis meliae with a query coverage at 100%. To fulfill Koch's postulates, pathogenicity tests were conducted on 30-year-old 'Lisbon' (Citrus x limon (L.) Osbeck) lemon trees at the Yuma Agriculture Center Mesa farm from February to July in 2019: ten branches (6-to-10 cm in diameter) from ten different trees were inoculated with each of three representative isolates. The inoculum was prepared by growing each isolate on wheat grains for three weeks at 23 to 25oC. Tree branches were inoculated by inserting five colonized wheat grains in 2-cm holes which were drilled into the branches and then covered with Parafilm. Five branches were inoculated with sterile grains and used as a control. Disease was assessed four months later by removing inoculated branches, splitting them in half through the inoculation point, and measuring the length of the wood area affected by wood rot. Brown discoloration of the wood extended an average of 3.9, 4.2, and 4.9 cm per isolate into wood tissues surrounding the inoculation hole, while control branches remained healthy. Fomitopsis meliae was consistently re-isolated from decayed wood based on morphology and ITS sequences. To our knowledge, this is the first report of brown wood rot caused by Fomitopsis meliae in lemon in Arizona and California. Fomitopsis species often attack conifers, hardwoods, and fruit trees (Adaskaveg et al., 1993, Gilbertson & Ryvarden, 1987). A species of Fomitopsis was detected in 40-year-old sweet orange trees and was highly pathogenic on lemons in southern Italy (Roccotelli et al., 2014). The ITS sequence of this Fomitopsis species (HM126455.1) shared 99% identity with those of Fomitopsis palustris (KJ995920.1) or F. ostreiformis (KC595918.1), but only 93.2% identity with that of F. meliae isolates identified in this study. Fomitopsis meliae can cause substantial pre-mature mortality of lemon trees and represents a major threat to the survival and profitability of lemon production in Arizona.

First Report of Crown and Root Rot Caused by Pythium aphanidermatum on Industrial Hemp (Cannabis sativa) in Arizona.

During July and August 2020, symptoms of leaf yellowing and browning, sudden wilting, and death were observed on industrial hemp plants (Cannabis sativa L.) in several drip-irrigated fields in Yuma and Graham county, Arizona. About 85% of plants showed severe crown and root rot symptoms. A high percentage of affected plants collapsed under intensive heat stress. Shriveled stem tissue with necrotic lesions can often be seen at the base of the plant, extending upwards more than 5 cm. Internal tissue of main stem and branches was darkened or pinkish brown. Outer cortex of root bark was often completely rotten, exposing the white core. Cottony aerial mycelium was visible on the surface of stalk of some of the infected plants in two fields in Yuma. To identify the causal agent, a total of twenty symptomatic plants were collected from several fields across the state. Crown and root tissues from affected plants were harvested and rinsed in tap water to remove soils. Approximately 2 to 4 mm tissue fragments were excised from the margins of the affected stem and root lesions, surface sterilized in 0.6% sodium hypochlorite for 1 min, rinsed copiously in sterile distilled water, blotted dry, and plated on potato dextrose agar (PDA), and on oomycete-selective clarified V8 medium containing pimaricin, ampicillin, rifampicin, and pentachloronitrobenzene (PARP). Plates were incubated at room temperature for 2 days. Sixteen isolates were recovered and their mycelial colonies resembled the morphology of Pythium. Based on the culture morphology on V8 medium, all isolates were tentatively identified as P. aphanidermatum with fast-growing, aseptate hyphae ranging from 3 to 7 µm in width, globose oogonia ranging from 25 to 31 µm in diameter, barrel-shaped antheridia, globose oospores ranging from 15 to 21 µm in diameter (10 measurements) (Watanabe, 2002). Genomic DNA was extracted from mycelial mats of three isolates using DNeasy Plant Pro Kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. The internal transcribed spacer (ITS) region of rDNA was amplified with primers ITS1/ITS4 and three nucleotide sequences were obtained. All three sequences were identical and deposited under accession number MW380253 in GenBank. A BLASTn search revealed that MW380253 had a 100% query coverage and 100% match with sequences MK611609.1, KJ162355.1, and AY598622.2, obtained from isolates of P. aphanidermatum. To fulfill Koch's postulates, pathogenicity tests were conducted with 2 isolates using 12 seeds of a hemp line 14 sown in 12 1.9-liter pots filled with a steam-disinfested potting mix. Pots were placed in a plastic container and watered three times a week by flooding, to create waterlogged conditions. Plants were maintained in a greenhouse supplemented with artificial lighting of 14 h/10 h day/night light cycle. Plants were fertilized weekly with a 20-20-20 fertilizer at 1mg/ml. Three weeks after sowing, four plants were inoculated with each isolate by drenching each plant with 200 ml of a 1×105 zoospore/ml suspension. Four plants, serving as control, received each 200 ml of distilled water. Symptoms of leaf chlorosis, crown and root rot, and wilting were observed 3 weeks afterwards, while control plants remained asymptomatic. P. aphanidermatum were re-isolated from necrotic roots of inoculated plants, but not from control plants. P. aphanidermatum was previously detected on industrial hemp in a research plot in Indiana (Beckerman et al., 2017) and is also known to affect other crops in Arizona during the summer months as well (Olsen & Nischwitz, 2011). This report is the first publication documenting P. aphanidermatum on field grown hemp in Arizona. Industrial hemp (Cannabis sativa) is an emerging crop in Arizona. The first plantings of hemp were in June of 2019, where 5,430 acres of hemp was planted in thirteen counties in Arizona before the end of the year. The Arizona Department of Agriculture Industrial Hemp Program, 2019 Year End Report confirms that nearly one-quarter of all hemp planted in 2019 did not receive a final state inspection due to crop loss. This disease is a potential constraint to hemp production in hot, arid climates, where copious water is used in combination with plastic mulch and/or drainage is poor.

First Report of Beet Curly Top Virus Infecting Industrial Hemp (Cannabis sativa) in Arizona.

Industrial hemp (Cannabis sativa) is an emerging crop in Arizona, with many uses, including fiber, cosmetic products, and health food. In 2020, severe curly top disease outbreaks were observed in several hemp fields in Yuma and Graham Counties, Arizona, where disease incidence and severity were considerably high, up to 100% crop loss occurring in some fields. A wide range of symptoms have been observed at different infection stages and plant growth stages at the time of infection. Early stage symptoms manifest as light green-to-yellowing of new growth, similar to sulfur or micronutrient deficiency, usually combined with older leaves with dark green "blotchy" mosaic mottling overlaying light green chlorosis. Mosaic mottling of older leaves continues into mid-growth stage, and is coupled with more severe yellowing and witch's broom (stunted leaves and shortened internode length of stem) of apical meristematic tissue. Curling and twisting of new leaves has also been observed. Symptoms often appear to be isolated to individual branches, with other branches showing no visual symptoms, often outgrowing and covering affected branches until harvest. Late stage symptoms include severe leaf curling with or without twisting, continued stunting, and necrosis of yellow leaves, resulting in significant yield reduction. Severely affected plants dwarfed by the virus experienced high mortality rates later into the season, most likely attributed to reduced ability to overcome abiotic stress conditions. These symptoms indicated the likelihood of curly top caused by Beet curly top virus (BCTV), which has been recently reported in Colorado (Giladi et al., 2020). Shoots were collected from thirty-eight symptomatic and nine asymptomatic hemp plants from July to August, 2020. Leaves were also collected as positive control from four chili pepper plants with or without curly top symptoms in Cochise County. Genomic DNA was extracted using DNeasy Plant Pro Kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. BCTV-specific primers BCTV1 and BCTV2 were used to detect BCTV following a method by Rondon (Rondon et al., 2016). A 500 bp DNA fragment, indicative of BCTV, was amplified from all symptomatic hemp and chili pepper samples, but not from asymptomatic samples. Sequence analysis of this 500 bp DNA fragment revealed 98.99 % identity with GenBank accession MK803280, which is Beet curly top virus isolate from hemp identified in Western Colorado (Giladi et al., 2020). The full-length genomes of BCTV isolates from hemp and chili peppers were generated with additional primers 328F/945R (620bp), 455F/ 945R (490bp), OutR/ 2213F (1,190bp), 2609R/ 1278R (1,340bp), BCTV2/ 2609R (1,890bp) (Rondon et al., 2016, Strausbaugh et al., 2008). The complete nucleotide sequence (MW182244) from hemp was 2,929 bp and had 99.35% sequence identity with GenBank accession KX867055, which was a Worland strain of Beet curly top virus isolated from an Idaho sugar beet plant (Strausbaugh et al., 2017). Our hemp BCTV genome sequences shared 96.08% identity with the hemp strain of BCTV from Colorado (MK803280) and 99.50% identity with the BCTV isolate (MW188519) from chili pepper identified in this study. BCTV was reported on outdoor hemp in Western Colorado, in 2020 (Giladi et al., 2020). This is the first report of BCTV in Arizona causing curly top of industrial hemp in the field. In Arizona, BCTV is widespread on many agronomic crops including chili peppers and spread primarily by the phloem-feeding beet leafhoppers: Circulifer tenellus (Hemiptera: Cicadellidae) (Bennett, 1967). Due to the wide distribution of beet leafhoppers and abundant range of host plants for the virus, BCTV may become one of the most yield-limiting factors affecting the emerging industrial hemp production systems in Arizona.

Evaluation of the Spatiotemporal Dynamics of Oxytetracycline and Its Control Effect Against Citrus Huanglongbing via Trunk Injection.

Citrus huanglongbing (HLB) or greening is a devastating bacterial disease that has destroyed millions of trees and is associated with phloem-residing 'Candidatus Liberibacter asiaticus' (Las) in Florida. In this study, we evaluated the spatiotemporal dynamics of oxytetracycline in planta and its control effect against HLB via trunk injection. Las-infected 'Hamlin' sweet orange trees on 'Swingle' citrumelo rootstock at the early stage of decline were treated with oxytetracycline hydrochloride (OTC) using trunk injection with varying number of injection ports. Spatiotemporal distribution of OTC and dynamics of Las populations were monitored by high-performance liquid chromatography method and qPCR assay, respectively. Uniform distribution of OTC throughout tree canopies and root system was achieved 2 days postinjection. High levels of OTC (>850 µg/kg) were maintained in leaf and root for at least 1 month and moderate OTC (>500 µg/kg) persisted for more than 9 months. Reduction of Las populations in root system and leaves of OTC-treated trees were over 95% and 99% (i.e., 1.76 and 2.19 log reduction) between 2 and 28 days postinjection. Conditions of trees receiving OTC treatment were improved, fruit yield was increased, and juice acidity was lowered than water-injected control even though their differences were not statistically significant during the test period. Our study demonstrated that trunk injection of OTC could be used as an effective measure for integrated management of citrus HLB.

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

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

UV-LASER adjuvant-surfactant-facilitated delivery of mobile dsRNA to tomato plant vasculature and evidence of biological activity by gene knockdown in the potato psyllid.

BACKGROUND: Double-stranded RNA (dsRNA) biopesticides are of interest for the abatement of insect vectors of pathogenic bacteria such as 'Candidatus Liberibacter', which infects both its psyllid and plant hosts. Silencing of genes essential for psyllids, or for Liberibacter, is anticipated to lead to mortality or impeded bacterial multiplication. Foliar delivery is preferred for biopesticide application; however, the cuticle impedes dsRNA penetration into the vasculature. Here, conditions were established for wounding tomato leaves using ultraviolet light amplification by stimulated emissions of radiation (UV-LASER) to promote dsRNA penetration into leaves and vasculature. RESULTS: UV-LASER treatment with application of select adjuvants/surfactants resulted in vascular delivery of 100-, 300- and 600-bp dsRNAs that, in general, were correlated with size. The 100-bp dsRNA required no pretreatment, whereas 300- and 600-bp dsRNAs entered the vasculature after UV-LASER treatment only and UV-LASER adjuvant/surfactant treatment, respectively. Of six adjuvant/surfactants evaluated, plant-derived oil combined with an anionic organosilicon compound performed most optimally. Localization of dsRNAs in the tomato vasculature was documented using fluorometry and fluorescence confocal microscopy. The biological activity of in planta-delivered dsRNA (200-250 bp) was determined by feeding third-instar psyllids on tomato leaves post UV-LASER adjuvant/surfactant treatment, with or without psyllid cdc42- and gelsolin dsRNAs. Gene knockdown was quantified by quantitative, real-time polymerase chain reaction with reverse transcription (RT-qPCR) amplification. At 10 days post the ingestion-access period, knockdown of cdc42 and gelsolin expression was 61% and 56%, respectively, indicating that the dsRNAs delivered to the tomato vasculature were mobile and biologically active. CONCLUSION: Results indicated that UV-LASER adjuvant/surfactant treatments facilitated the delivery of mobile, biologically active dsRNA molecules to the plant vasculature. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Mefenoxam Sensitivity in Phytophthora cinnamomi Isolates.

Phytophthora cinnamomi is a destructive root pathogen of numerous woody plant species in the ornamental plant nursery. Sixty-five isolates of P. cinnamomi were evaluated for mefenoxam sensitivity on 20% clarified V8 agar amended with mefenoxam at 0 or 100 µg/ml. In the presence of mefenoxam at 100 µg/ml, eight isolates were intermediately sensitive, with mycelium growth ranging between 11 and 18% of the nonamended control, and 57 isolates were highly sensitive, with little or no mycelium growth. Five intermediately sensitive and five sensitive isolates were chosen to characterize their responses to mefenoxam at 0, 0.1, 1, 10, and 100 µg/ml. For intermediately sensitive isolates, the mefenoxam concentration causing 50% inhibition of mycelium growth (EC50 values) ranged between 0.03 and 0.08 µg/ml; EC50 values for sensitive isolates varied from 0.01 to 0.02 µg/ml. Five intermediately sensitive and seven sensitive isolates were selected further to assess in vivo sensitivity to mefenoxam using Lupinus angustifolius 'Russell Hybrids'. Lupine seedlings were treated with distilled water or mefenoxam at label rate (Subdue MAXX, 1 fl. oz. of product per 100 gal.) and then, 2 days later, inoculated with a 5-mm-diameter mycelial plug of P. cinnamomi on each cotyledon. Mefenoxam-treated plants averaged more than 96% less disease than water-treated plants. Mefenoxam provided adequate protection of lupines from infection by all 12 isolates regardless of their in vitro levels of sensitivity to mefenoxam. The ability to develop mefenoxam resistance was assessed in P. cinnamomi isolates with different mefenoxam sensitivity by UV mutagenesis and adapting mycelium to increasing concentrations of mefenoxam. Both UV mutagenesis and mycelium adaptation generated isolates with reduced sensitivity to mefenoxam. These isolates, however, did not grow as quickly as their corresponding parent. This study suggests that P. cinnamomi populations from ornamental nurseries in Virginia are sensitive to mefenoxam.

Effects of Propamocarb Hydrochloride on Mycelial Growth, Sporulation, and Infection by Phytophthora nicotianae Isolates from Virginia Nurseries.

Propamocarb hydrochloride is a systemic fungicide commonly used for control of Phytophthora diseases of nursery crops. Here we report on the effect of this compound on different growth stages of Phytophthora nicotianae, a major pathogen of numerous herbaceous and some woody ornamental plants. A total of 71 isolates were assayed for sensitivity to propamocarb at two concentrations of 1.8 mg/ml (label rate) and 10 mg/ml using clarified V8 agar as a base medium. All isolates grew at 10 mg/ml with the most sensitive isolate having 34.8% relative growth compared with growth on nonamended medium. Nine isolates were selected and further tested for mycelial growth at 0, 1, 10, 25, 50, and 100 mg/ml, and for sporangium production, zoospore motility, and germination at 0, 5, 50, 500, 5,000, and 50,000 µg/ml. EC50 values ranged from 2.2 to 90.1 mg/ml for mycelial growth, 133.8 to 481.3 µg/ml for sporangium production, 88.1 to 249.8 µg/ml for zoospore motility, and 1.9 to 184.6 µg/ml for zoospore germination, respectively. In addition, 17 selected isolates were evaluated for propamocarb sensitivity on Pelargonium × hortorum cv. White Orbit. Two days after seedlings were treated with propamocarb at 3.6 mg/ml, they were inoculated by either inverting one 5-mm-diameter plug of a 3-day-old culture or applying a 10-µl drop containing 20 zoospores onto each cotyledon. Propamocarb hydrochloride provided good protection of geranium seedlings from zoospore infections but not from mycelial infections. These results suggest that this fungicide must be used preventively for Phytophthora disease management and that mycelial growth may not be the most reliable measurement to determine the development of fungicide resistance to this compound in Phytophthora species at production facilities and in the landscape.

Development of a Microemulsion Formulation for Antimicrobial SecA Inhibitors.

In our previous study, we have identified five antimicrobial small molecules via structure based design, which inhibit SecA of Candidatus Liberibacter asiaticus (Las). SecA is a critical protein translocase ATPase subunit and is involved in pre-protein translocation across and integration into the cellular membrane in bacteria. In this study, eleven compounds were identified using similarity search method based on the five lead SecA inhibitors identified previously. The identified SecA inhibitors have poor aqueous solubility. Thus a microemulsion master mix (MMX) was developed to address the solubility issue and for application of the antimicrobials. MMX consists of N-methyl-2-pyrrolidone and dimethyl sulfoxide as solvent and co-solvent, as well as polyoxyethylated castor oil, polyalkylene glycol, and polyoxyethylene tridecyl ether phosphate as surfactants. MMX has significantly improved the solubility of SecA inhibitors and has no or little phytotoxic effects at concentrations less than 5.0% (v/v). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the SecA inhibitors and streptomycin against eight bacteria including Agrobacterium tumefaciens, Liberibacter crescens, Rhizobium etli, Bradyrhizobium japonicum, Mesorhizobium loti, and Sinorhizobium meliloti phylogenetically related to Las were determined using the broth microdilution method. MIC and MBC results showed that the 16 SecA inhibitors have antibacterial activities comparable to that of streptomycin. Overall, we have identified 11 potent SecA inhibitors using similarity search method. We have developed a microemulsion formulation for SecA inhibitors which improved the antimicrobial activities of SecA inhibitors.

Survival of Salmonella enterica Typhimurium in water amended with manure.

Outbreaks of Salmonella enterica have been associated with water sources. Survival of S. enterica in various environments has been studied but survival in water has rarely been attempted. In two separate experiments, we examined the survival of S. enterica Typhimurium in clean spring water at various eutrophication levels and temperatures. In the first experiment, lasting for 135 days, survival of S. enterica (10(10) CFU/ml) in water with 0, 50, 100, 500, and 1,000 mg/liter of added carbon at 7, 17, and 27°C was monitored weekly. In the second experiment, lasting for 3 weeks, survival of S. enterica in water at 0, 100, and 200 mg/ liter of added carbon and 27°C was studied daily. Each experiment had four replicates. Dissolved organic carbon was measured daily in each experiment. At the beginning, midpoint, and end of the survival study, microbial communities in both experiments were assessed by denaturing gradient gel electrophoresis (DGGE). Even at minimal carbon concentrations, S. enterica survived for at least 63 d. Survival of Salmonella was highly dependent on eutrophication levels (as measured by dissolved organic carbon) and temperature, increasing at high eutrophication levels, but decreasing at high temperatures. Survival was also strongly affected by microbial competition or predation.

Assessment of peanut quality and compositional characteristics among transgenic sclerotinia blight-resistant and non-transgenic susceptible cultivars.

This study presents the results of a comparison that includes an analysis of variance and a canonical discriminant analysis to determine compositional equivalence and similarity between transgenic, sclerotinia blight-resistant and non-transgenic, susceptible cultivars of peanut in 3 years of field trials. Three Virginia-type cultivars (NC 7, Wilson, and Perry) and their corresponding transgenic lines (N70, W73, and P39) with a barley oxalate oxidase gene were analyzed for differences in key mineral nutrients, fatty acid components, hay constituents, and grade characteristics. Results from both analyses demonstrated that transgenic lines were compositionally similar to their non-transgenic parent cultivar in all factors as well as market-grade characteristics and nutritional value. Transgenic lines expressing oxalate oxidase for resistance to sclerotinia blight were substantially equivalent to their non-transgenic parent cultivar in quality and compositional characteristics.