A Quantitative Real-Time PCR Method to Detect the Quinoa Downy Mildew Pathogen, Peronospora variabilis.

Quinoa downy mildew, caused by Peronospora variabilis, is the most devastating disease of quinoa globally. Rapid, sensitive diagnostic methods are needed to detect and quantify this pathogen in seeds and plant tissue. A hydrolysis probe-based quantitative real-time PCR (qPCR) assay including a competitive internal control was developed for P. variabilis detection. This assay could detect as low as 20 ag of DNA or approximately 25 internal transcribed spacer (ITS) copies per reaction with efficiencies ranging from 93.9 to 98.2%. No non-target amplification was observed when tested against DNA from other downy mildew pathogens and related oomycetes. Peronospora variabilis strains from multiple countries were detected using this assay. The assay was successfully applied to quantify the pathogen in quinoa seeds from a field trial conducted in Washington State. Downy mildew disease was recorded on all 14 genotypes with the genotypes 104.88 and 106.49 recording the highest area under the disease progress values (3,236 ± 303 SE and 2,851 ± 198, respectively) while J6 and Dutchess recorded the lowest (441 ± 107 and 409 ± 129, respectively). Seed washes obtained from field samples were subjected to the qPCR assay, and the pathogen was detected in all samples. The highest pathogen ITS copy number recorded with 106.49 (194,934 ± 38,171 SE), while the lowest was observed in Pasto (5,971 ± 1,435) and Riobamba (9,954 ± 4,243). This qPCR assay could lead to improved detection and quantification of P. variabilis as well as increased understanding of quinoa-P. variabilis interactions and epidemiology.

First Report of Diaporthe fruit rot and stem canker of hydroponic cucumber caused by Diaporthe sp. in the United States.

Multiple Diaporthe spp. cause root and fruit rots or stem lesions on Cucumis spp.: D. cucurbitae, D. melonis, D. longicolla (syn. D. eres), D. pterocarpi, D. sclerotioides, D. sojae, and D. ueckerae (Broge et al. 2020; Fukada et al. 2018; Udayanga et al. 2012, 2015). From May-August 2021, cucumbers (Cucumis sativus) 'Katrina' and 'Alcazar' were grown in a 24-plant, commercial Bato bucket system with rockwool blocks on a perlite substrate in a research greenhouse in Wooster, Ohio. At maturity, plants collapsed rapidly from stem lesions without foliar chlorosis (25% of 'Katrina' and 17% of 'Alcazar'). Lesions were 7.5 to 15 cm in length, tan to golden-brown with black pycnidia and located 5 to 15 cm above the crown. Stems shredded easily with vascular discoloration around the lesion. Two identical fungal strains were isolated on ½ acidified potato dextrose agar (APDA) following surface disinfestation with 0.6% sodium hypochlorite for 30 s and sterile water rinse. Fungal cultures were floccose, white to tan mycelia with pycnidia. Oblong, elliptical, biguttulate, aseptate alpha conidia were observed with mean dimensions: 8.0 µm (5.2-9.8 µm) by 3.1 µm (2.5-3.8 µm) on ½ APDA and 9.8 µm (6.6-12.4 µm) by 3.0 µm (1.9-5.3 µm) on petioles. On prune extract agar, beta conidia mean dimensions were: 19.7 µm (12.0-27.7 µm) by 1.2 µm (0.8-1.8 µm). Fungal DNA was amplified and sequenced bidirectionally with ITS (ITS4/ITS5), CAL (CAL228F/737R), HIS (CYLH3F/H3-1B), TEF1 (EF1-728f/EF1-986R), and TUB2 (Bt1a/Bt1b) primers (Carbone and Kohn 1999; Glass and Donaldson 1995) (GenBank: OP265712-13, OP288460-65, OQ418506-07). Based on a maximum likelihood phylogenetic tree of concatenated genes, this novel Diaporthe sp., most closely related to D. stewartii, has not been reported on Cucumis spp. Strains were deposited in the USDA-ARS Culture Collection (NRRL# 64461-62). Koch's postulates were conducted in a greenhouse with mean day temperature of 25°C and 12 hr supplemental lighting. One-month old cucumbers 'Katrina,' grown in rockwool cubes (5 plants per isolate) and potting mix (6 plants per isolate), were inoculated with a one-week-old culture of either strain. The second true leaf was cut and a pipette tip containing an inoculated plug of ½ APDA was placed on the remaining petiole (Mathew et al. 2018). Non-inoculated ½ APDA was used for controls. Plants were tarped for 24 hours to increase humidity and pipette tips removed after one week. After two weeks, petioles were shrunken, tan to golden brown with pycnidia. After 3-4 weeks, stem lesions matching those above were observed on inoculated plants, and plants collapsed. For fruit rot, three Beit Alpha cucumbers were rinsed with tap water, dried, a 5 mm plug was removed from the fruit and replaced with a 5 mm plug of one-week-old fungus on ½ APDA. After 3 days, fruits were water soaked and soft. For root rot, two plates of one-week-old cultures were macerated in 500 mL of sterile water and mixed with 1500 mL of vermiculite. Two seeds of cucumber 'Katrina' were planted into three reps of each isolate and control. All control seeds germinated, but all inoculated seeds experienced pre- or post-emergence damping off. No symptoms were ever observed on any controls. Fungi were isolated from all inoculated tissues as described above. Based on morphology, Diaporthe sp. was isolated from all inoculated plants but never from controls. This Diaporthe sp. may be a new constraint to hydroponic cucumber production, but incidence needs to be determined globally.

First report of quinoa anthracnose caused by Colletotrichum nigrum and C. truncatum in the United States.

Quinoa (Chenopodium quinoa Willd.) is increasingly produced outside its native Andean range. In September 2019, stem lesions were observed on all six plants of quinoa accessions PI 510547 (25% severity) and PI 596293 (75% severity) in a demonstration plot in Ames, IA. Lesions were bleached, silvery-white to dark gray, slightly sunken, oval to linear with slightly tapered tips and contained setose acervuli. Fungi were isolated from both accessions following disinfestation with 70% ethanol and plating onto ½ acidified potato dextrose agar (APDA) and V8 medium. Isolates were examined morphologically. On V8 medium, isolate CQ1 produced sparse, flat, gray mycelia with profuse sclerotia and hyaline, aseptate, cylindrical conidia (n= 50, mean: 21.0 (range: 19.2-24) by 4.3 (2.4-4.8) µm); isolate CQ2 produced fluffy, gray to dark gray mycelia with profuse sclerotia and acervuli and hyaline, aseptate, falcate conidia (n =50, 26.8 (24-31.2) by 2.4 µm). Direct hyphal PCR was used to amplify ITS (ITS1/ITS4), ACT (ACT-512F/ACT-783R), GAPDH (GDF1/GDR1), CHS-1 (CHS-79F/CHS-234R), and TUB2 (T1/Bt-2b, CQ1 only) (Fu et al. 2019, Liu et al. 2013), and products were sequenced bidirectionally (MT772082-3, MT786524-30). A maximum likelihood tree was generated in MEGA X (Kumar et al. 2018) from a multiple sequence alignment of vouchered CBS isolates (Liu et al. 2013) and CQ1 was identified as Colletotrichum nigrum. CQ2 sequences showed 99-100% similarity to Colletotrichum truncatum sequences in Genbank (MN581860, MK675238, MF682518, MK118057). Koch's postulates were completed once with two isolates of each species grown on V8 medium under 12 hours of near UV light for two weeks. Greenhouse conditions were a 12 hr day/night cycle and temperature range of 26-30° C. Approximately 5 mL of mycelium on agar medium was sterilely removed and macerated in 6 mL of sterile distilled water. Non-inoculated medium was macerated in sterile water as a control. Forty-day old quinoa PI 634920 were inoculated by making three, 2-3 mm incisions between the cotyledons and first true leaves with a sterile razor blade. Next, 500 µL of slurry was placed on 2.54 cm2 of sterile cheesecloth and placed against the wound and wrapped with Parafilm. Six plants were inoculated per isolate and control. After two weeks, sunken, bleached to tan areas extended past wound sites of inoculated plants. No discoloration or sunken tissue was observed on control plants. Plants were tented with plastic film for one week. Acervuli were observed on C. truncatum- and C. nigrum-inoculated stems, and sclerotia were observed on C. nigrum-inoculated stems. Stems were surface disinfested with 10% bleach and plated onto ½ APDA. Colony morphologies of isolated fungi matched those of original inoculum for inoculated plants. Colletotrichum spp. were never isolated from control plants. When stems were inoculated, approximately 100 µL of slurry was also placed on 3-5 detached quinoa leaves in Petri dishes with moistened blotter paper and incubated for 48 hours at 25° C. Brownish, circular lesions developed on leaves inoculated with either species, but no lesions developed on control-slurry leaves. Colletotrichum spp. cause disease in quinoa relatives including spinach (Kurt 2015), beets (Gourley 1966) and amaranth (Wu 2001). This is the first description of Colletotrichum spp. causing stem lesions on quinoa in the United States. This disease may emerge in new quinoa production regions and may cause yield losses due to lodging.

First report of Xanthomonas hortorum causing bacterial leaf spot of lavender (Lavandula × intermedia) in Ohio.

In July 2018, a sample of lavender var. Grosso (Lavandula × intermedia 'Grosso') from Miami County, OH was received by The Ohio State University Vegetable Pathology Laboratory in Wooster. Lavender plants were field-grown in sandy clay soil with plastic mulch under drip irrigation. Disease incidence ranged from 0 to 32% depending on variety. Leaves and stems showed dark necrotic lesions that varied from roughly circular (ca. 0.3 to 0.5 mm diameter) to large coalesced necrotic areas surrounded by a water-soaked halo. Bacterial streaming from lesions was observed microscopically. Leaf tissue pieces (~0.5 cm2) were surface sterilized in 70% ethanol for 30 seconds and rinsed in sterile deionized water. The tissue was sliced aseptically into smaller sections in 100 µl sterile water and the bacterial suspension was streaked on yeast dextrose calcium carbonate agar medium. Ten yellow Xanthomonas-like colonies were selected after 72 hours of incubation at 28ºC in the dark. Strains were gram negative, oxidase negative and caused hypersensitive reactions on Nicotiana benthamiana (L.). All strains were genotyped after whole-cell DNA extraction by BOX-PCR (Louws et al. 1999) and had the same banding profile. Four 8-wk-old lavender plants (Lavandula dentata and Lavandula × ginginsii 'Goodwin Creek Gray') were spray-inoculated with a 106 CFU/ml suspension of strain SM175-2018 in sterile water. Control plants were sprayed with sterile water. Plants were kept in plastic bags for the first 48 h at 28°C with a 14-h photoperiod. Water-soaked necrotic lesions appeared 14 days after inoculation with SM175-2018, whereas mock-inoculated plants did not show symptoms. Bacterial isolation from symptomatic leaf tissue was carried out as described above. The BOX-PCR profile of the re-isolated strain was identical to that of SM175-2018. Multilocus sequence analysis of the housekeeping genes fuyA, gyrB, and rpoD was performed (Accession numbers: MT764834 - MT764836). The resulting concatenated data set was used to perform a phylogenetic analysis using maximum likelihood criteria to evaluate relationships with closely related Xanthomonas spp. using published reference sequences (Young et al. 2008). SM175-2018 was assigned to the X. hortorum clade (Moriniere et al. 2020) with strong bootstrap support. The strain was subjected to whole genome analysis. Genomic DNA was extracted using a QIAGEN Genomic DNA buffer set with genomic-tip 100/G following manufacturer's protocol and sequenced using the iSeq-100 Illumina platform with the Nextera DNA Flex Library Prep protocol kit and Nextera DNA CD indexes. Average nucleotide identity (ANI) analysis was performed with the ANI-Matrix software Enveomics tool (Rodriguez-R and Konstantinidis 2016) using the sequenced genome (NCBI GenBank Biosample no. SAMN11831455) and those of other X. hortorum (Vauterin et al. 1995) bacteria (pvs. hederae, carotae, vitians). SM175-2018 shared a 96% ANI with other X. hortorum strains. X. hortorum is associated with bacterial leaf spot of carrot (Scott and Dung, 2020) and also reported on ornamental plants (Mirik et al. 2010, Oliver et al. 2012, Roberts and Parkinson 2014, Klass et al. 2019), however additional research is needed to establish the host specificity of lavender strains. To our knowledge this is the first report of X. hortorum causing bacterial leaf spot of lavender in Ohio. The disease may negatively impact the yield and quality of flowers used in production of lavender oils and essences.