Crown Rot Symptoms Research Articles

First report of Fusarium commune causing root and crown rot on maize in Italy.

Maize (Zea mays L.) is a cereal crop of great economic importance in Italy; production is currently of 62,587,469 t, with an area that covers 628,801 ha, concentrated in northern Italy (ISTAT 2020). Fusarium species are associated with root and crown rot causing failures in crop establishment under high soil moisture. In 2019 maize seedlings collected in a farm located in San Zenone degli Ezzelini (VI, Italy) showed root and crown rot symptoms with browning of the stem tissues, wilting of the seedling, and collapsing due to the rotting tissues at the base of the stem. The incidence of diseased plants was approximately 15%. Seedlings were cleaned thoroughly from soil residues under tap water. Portions (about 3-5 mm) of tissue from roots and crowns of the diseased plants were cut and surface disinfected with a water solution of NaClO at 0.5% for 2 minutes and rinsed in sterile H20. The tissue fragments were plated on Potato Dextrose Agar (PDA) amended with 50 mg/l of streptomycin sulfate and incubated for 48-72 hours at 25oC. Over the 80 tissue fragments plated, 5% were identified as Fusarium verticillioides, 60% as Fusarium spp., 35% developed saprophytes. Fusarium spp. isolates that showed morphological characteristics not belonging to known pathogenic species on maize were selected and used for further investigation while species belonging to F. oxysporum were discarded. Single conidia of the Fusarium spp. colonies were cultured on PDA and Carnation Leaf Agar (CLA) for pathogenicity tests, morphological and molecular identification. The colonies showed white to pink, abundant, densely floccose to fluffy aerial mycelium. Colony reverse showed light violet pigmentation, in rings on PDA. On CLA the isolates produced slightly curved macronidia with 3 septa 28.1 - 65.5 µm long and 2.8-6.3 µm wide (n=50). Microconidia were cylindrical, aseptate, 4.5 -14.0 µm long and 1.5-3.9 µm wide (n=50). Spherical clamydospores were 8.8 ± 2.5 µm size (n=30), produced singly or in pairs on the mycelium, according to the description by Skovgaard et al. (2003) for F. commune. The identity of two single-conidia strains was confirmed by sequence comparison of the translation elongation factor-1α (tef-1α), and RNA polymerase II subunit (rpb2) gene fragments (O'Donnell et al. 2010). BLASTn searches of GenBank, and Fusarium-ID database, using the partial tef-1α (MW419921, MW419922) and rpb2 (MW419923, MW419924) sequences of representative isolate DB19lug07 and DB19lug20, revealed 99% identity for tef-1α and 100% identity to F. commune NRRL 28387(AF246832, AF250560). Pathogenicity tests were carried out by suspending conidia from a 10-days old culture on PDA in sterile H2O to 5×104 CFU/ml. Fifty seeds were immersed in 50 ml of the conidial suspension of each isolate for 24 hours and in sterile water (Koch et al. 2020). The seeds were drained, dried at room temperature, and sown in trays filled with a steamed mix of white peat and perlite, 80:20 v/v, and maintained at 25°C and RH of 80-85% for 14 days with 12 hours photoperiod. Seedlings were extracted from the substrate, washed under tap water, and observed for the presence of root and crown rots like the symptoms observed on the seedlings collected in the field. Control seedlings were healthy and F. commune was reisolated from the symptomatic ones and identified by resequencing of tef-1α gene. F. commune has been already reported on maize (Xi et al. 2019) and other plant species, like soybean (Ellis et al. 2013), sugarcane (Wang et al. 2018), potato (Osawa et al. 2020), indicating that some attention must be paid in crop rotation and residue management strategies. To our knowledge this is the first report of F. commune as a pathogen of maize in Italy. References Ellis M L et al. 2013. Plant Disease, 97, doi: 10.1094/PDIS-07-12-0644-PDN. ISTAT. 2020. http://dati.istat.it/Index.aspx?QueryId=33702. Accessed December 28, 2020. Koch, E. et al. 2020. Journal of Plant Diseases and Protection. 127, 883-893 doi: 10.1007/s41348-020-00350-w O'Donnell K et al. 2010. J. Clin. Microbiol. 48:3708. https://doi.org/10.1128/JCM.00989-10 Osawa H et al. 2020. Journal of General Plant Pathology, doi.org/10.1007/s10327-020-00969-5. Skovgaard K 2003. Mycologia, 95:4, 630-636, DOI: 10.1080/15572536.2004.11833067. Wang J et al. 2018. Plant Disease, 102, doi/10.1094/PDIS-07-17-1011-PDN Xi K et al. 2019. Plant Disease, 103, doi/10.1094/PDIS-09-18-1674-PDN.

Co-inoculation of Trichoderma gamsii A5MH and Trichoderma harzianum Tr906 in wheat suppresses in planta abundance of the crown rot pathogen Fusarium pseudograminearum and impacts the rhizosphere soil fungal microbiome

Wheat root endophytic strains Trichoderma gamsii A5MH and T. harzianum Tr906 were assessed for their abilities to suppress in planta abundance of the crown rot pathogen Fusarium pseudograminearum and increase growth of durum wheat. Fusarium crown rot suppressive assays were conducted in natural Kandosol and Chromosol wheat cropping soils with greater disease expression observed in the Chromosol. Rhizosphere abundance of T. gamsii A5MH, T. harzianum Tr906 and F. pseudograminearum were quantified by species-specific qPCR at plant maturity. Strains A5MH and Tr906 actively colonized the wheat rhizosphere and suppressed in planta pathogen abundance in both soil types, with both inoculants persisting in roots and soil to 84 days postemergence. Strain A5MH increased wheat biomass in both soils, whereas strain Tr906 only increased biomass of Chromosol-grown wheat. Metabolites of strains A5MH and Tr906 decreased in vitro growth of wheat pathogenic F. pseudograminearum, Rhizoctonia solani and Pythium irregulare, but did not inhibit either inoculant. Compared with individually inoculated strains, co-inoculation of A5MH and Tr906 did not affect rhizosphere niche abundance, in planta pathogen suppressive efficacies or wheat growth-benefits of either inoculant in Chromosol-grown wheat. Fusarium crown rot symptoms however, only decreased in the A5MH and co-inoculated treatments. Each Trichoderma treatment had distinct impacts on the structure (beta diversity) of the indigenous rhizosphere soil fungal community of Chromosol-grown wheat. Strain A5MH and co-inoculated treatments had the greatest overall effects on soil fungal community (alpha) diversity and structure. Community differentiation was primarily attributed to increased abundance of Trichoderma spp. in the inoculated treatments and associated decreases in plant pathogenic and predominantly saprophytic fungal genera.

First Report of Crown Rot Caused by Fusarium redolens on Wheat in Kazakhstan.

Fusarium crown rot, caused by several species within the genus, is a major constraint that results in significant losses in wheat production worldwide. In June 2019, diseased wheat plants with typical symptoms of crown rot, including discoloration on the first two or three internodes of the stem just above the soil line and stunted, dry rotted, and discolored roots were collected in several bread wheat fields during the maturity stage in Almaty, East Kazakhstan, and Karaganda Regions of Kazakhstan. For each field, approximately twenty tillers were randomly sampled. Symptomatic tissues were surface sterilized in 1% NaClO for 2 min, rinsed with sterile distilled water three times, air-dried in a laminar flow hood, and then transferred to Petri dishes containing one-fifth strength potato dextrose agar (PDA). After incubating in the dark at 23°C for 5 days, 79 single-spore isolates showing cultural and microscopic characteristics of Fusarium were obtained on PDA and Spezieller-Nährstoffarmer agar (SNA). Colonies were initially white but later produced a beige to pink diffusible pigment in PDA. Microconidia that formed on aerial monophialides were hyaline, 0 to 1 septum, oval- to kidney-shaped, and measured 4.3 to 10.3 × 1.9 to 3.4 µm (average 7.8 × 2.6 µm), whilst macroconidia were straight to slightly curved, 3 to 5 septate, and measured 18.7 to 38.8 × 2.9 to 6.6 µm (average 29.9 × 4.7 µm), with foot-shaped basal cells on SNA. Chlamydospores were present on PDA. Sequence analysis based on portions of translation elongation factor 1α (TEF1) and the nuclear ribosomal internal transcribed spacer region (ITS rDNA) loci with primers EF1/EF2 (O'Donnell et al. 1998) and ITS1/ITS4 (White et al. 1990) identified 29 of the 79 isolates as Fusarium redolens Wollenw. The sequences of the five representative isolates with 99.85% of similarity to those of F. redolens strains available in GenBank e.g., ITS (MT435063) and TEF1 (GU250584). The TEF1 (accession nos. MW403914-MW403918) and ITS rDNA (accession nos. MW397138-MW397142) sequences of the isolates were deposited in GenBank. The morphological features are consistent with the described features of F. redolens (Leslie and Summerell 2006). To confirm pathogenicity of the five isolates, five pre-germinated seeds of wheat cultivar Seri 82 were placed in a 9-cm-diameter pot filled with a sterile potting mix containing equal volumes of peat, vermiculite, and soil. An approximately 1-cm-diameter 7-day-old mycelial plug of each isolate was individually placed in contact with the seeds. Seeds were covered with the same potting mix, and then the pots were maintained for four weeks in a growth chamber at 23°C with a 12-h photoperiod. The experiment was conducted twice with three replicate 15-cm pots with 5 plants per pot. Controls were inoculated with sterile agar plugs using the same procedure. After four weeks, all the inoculated plants showed stunted growth with brown discoloration in most parts of the crown and roots, whereas no symptoms were observed in the control plants. The mean severity of the disease for each isolate was between 2.1 and 2.7 according to the scale of 1 to 5 described by Gebremariam et al. (2015). The pathogen was reisolated from crowns of diseased plants, but not from asymptomatic control tissues, and identified morphologically based on the methods described above, fulfilling Koch's postulates. Although several morphological features are shared by F. oxysporum and F. redolens, Baayen et al. (2001) showed that these species could be easily distinguished using molecular data. The pathogen was previously reported as F. redolens associated with crown rot of wheat in Turkey (Gebremariam et al. 2015) and Saskatchewan, Canada (Taheri et al. 2011). The presence of F. redolens causing crown rot is confirmed in the six wheat fields surveyed in Kazakhstan, for the first time. This pathogen may pose a risk for wheat production, and further studies needed to determine the impact on the crop in Kazakhstan.

Several Pythium species cause crown and root rot on cannabis (Cannabis sativa L., marijuana) plants grown under commercial greenhouse conditions

Cannabis (Cannabis sativa L., marijuana) plants with symptoms of crown rot, root decay, wilting and plant death were sampled during 2018 and 2019 from seven production greenhouses. Affected tissues from 140 diseased plants were surface-sterilized and plated onto potato dextrose agar. Ninety-five isolates morphologically resembling Pythium species were subcultured and subjected to PCR of the ITS1-5.8-ITS2 region of ribosomal DNA. The following species were identified based on >99% sequence identity to reference isolates in GenBank: P. myriotylum (43 isolates), P. dissotocum (35 isolates), P. aphanidermatum (3 isolates) and Globisporangium ultimum (syn. P. ultimum) (2 isolates). A fifth species – P. catenulatum (12 isolates), was distinguished from P. rhizo-oryzae using the cytochrome oxidase c subunit I (COI) sequence. Cannabis licensed production facilities in British Columbia had all five species present, while P. dissotocum was found in two facilities in Ontario, and P. myriotylum was present in one facility in northern California. Isolates selected to represent each Pythium species were grown on potato dextrose agar at 25 oC and they all showed comparable colony growth after 6 days. The same isolates caused root browning, decay and stunting of cannabis plants grown in a coco: perlite potting medium. Plant mortality was similar after 21 days but rates of disease progression varied depending on the isolate tested. Wounding of roots and prolonged periods of saturation enhanced disease development. These results demonstrate for the first time that crown and root rot on greenhouse grown cannabis plants can be caused by up to five Pythium species.

Developing Actinobacterial Endophytes as Biocontrol Products for Fusarium pseudograminearum in Wheat.

Crown rot of wheat, caused by Fusarium pseudograminearum, results in millions of dollars of yield losses globally each year. Management strategies to control crown rot are limited and there are concerns about development of fungicide resistance so novel treatment strategies are desirable. A collection of endophytic Actinobacteria was screened for their ability to suppress the growth of F. pseudograminearum and the development of crown rot symptoms in wheat with the aim of identifying candidates that can be developed into biocontrol products. The ability of the Actinobacteria isolates to suppress the growth of three different F. pseudograminearum strains in vitro was assessed using agar-plate competition assays. Soil-free seedling assays were used to screen for suppression of development of early disease symptoms in the susceptible wheat (Triticum aestivum) cv. Tamaroi. Four of the isolates were tested in a glasshouse pot experiment to assess their ability to decrease disease symptoms and prevent yield losses in wheat cv. Tamaroi grown to maturity in an unsterilized soil. The screening of 53 isolates identified two Streptomyces isolates, MH71 and MH243, with very strong antifungal activity against F. pseudograminearum strains in agar-plate competition and seedling assays. In the glasshouse pot trial, plants treated with seed coatings of either MH71 or MH243 had > 24% lower disease severity than control plants infected with F. pseudograminearum. These two cultures show potential for development as biocontrol products because they are easy to culture, grow on relatively inexpensive media, produce highly durable spores and can be delivered to plants as a seed coat.

Screening of sugar beet pre-breeding populations and breeding lines for resistance to Ditylenchus dipsaci penetration and reproduction

Ditylenchus dipsaci is an economically important plant-parasitic nematode affecting European sugar beets. To date, no sugar beet cultivars carrying resistance against D. dipsaci are available to farmers. To find potentially resistant sugar beet lines restricting reproduction and penetration of D. dipsaci, three consecutive in vivo bioassays were carried out. The first experiment determined the penetration rate of D. dipsaci in 79 breeding lines and 14 pre-breeding populations. Based on these results, D. dipsaci penetration and reproduction resistance of eight genotypes was intensively investigated. It could be demonstrated that none of the genotypes showed resistance towards D. dipsaci. However, a high variation of the penetration rate by D. dipsaci was observed among the genotypes. The breeding line ‘DIT_119’ effectively reduced D. dipsaci penetration (34.4 ± 8.8 nematodes/plant at 22 days post-planting) compared to the susceptible control (109.0 ± 16.9) while ensuring a yield comparable to non-inoculated plants. However, the breeding line ‘DIT_119’ did not reduce D. dipsaci reproduction. The paternal line of the cultivar BERETTA KWS, demonstrating a high tolerance to D. dipsaci crown rot symptoms, did not reduce penetration and reproduction. Thus, no correlation can be established between reduced penetration rates, reproduction, and tolerance to D. dipsaci. This study provides an essential basis for the development of resistant sugar beet cultivars to D. dipsaci. The variations observed among genotypes now need to be confirmed with larger-scale screenings.

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.

Identification and Chemical and Biological Management of Phytopythium vexans, the Causal Agent of Phytopythium Root and Crown Rot of Woody Ornamentals.

Soilborne diseases caused by pathogens such as Phytophthora, Rhizoctonia, Fusarium, Verticillium, and Pythium species are the most important diseases of woody ornamentals. Ginkgo (Ginkgo biloba) and red maple (Acer rubrum 'October Glory') plants grown in containers and fields in Tennessee showed root and crown rot symptoms with dark brown to black lesions in 2017 and 2018. The objective of this research was to isolate and identify pathogens affecting ginkgo and red maple plants in Tennessee nurseries and to develop fungicide/biofungicide management recommendations for nursery producers. Isolations were made from the infected roots. Several Phytophthora-like colonies with spherical zoospores, filamentous to globose oogoni, and whitish mycelium were isolated on V8-PARPH medium. To confirm identity, total genomic DNA was extracted, followed by sequence analysis of the internal transcribed spacer regions, large subunit of nuclear rRNA, and cytochrome c oxidase subunits I and II of mitochondrial DNA. Based on morphological and molecular analysis, Phytopythium vexans was described as a causal agent of crown and root rot from the infected ginkgo and red maple plants. To complete Koch's postulates, a pathogenicity test was performed by drenching 100 ml of V8 agar medium slurry of Phytopythium vexans inoculum on 1-year-old potted ginkgo plant root systems as well as red maple October Glory. Necrotic lesion development was observed in the root system 45 days after inoculation and Phytopythium vexans was reisolated from the roots of both ginkgo and red maple. All control ginkgo and red maple plants remained disease free and no pathogen was reisolated. In addition, the efficacy of fungicides, biofungicides, fertilizer, and host plant defense inducers (traditionally recommended for management of oomycete diseases) for control of Phytopythium crown and root rot was evaluated on ginkgo and red maple October Glory seedlings in greenhouse and field trials. Fungicides such as Empress Intrinsic, Pageant Intrinsic, Segovis, and Subdue MAXX were effective in both greenhouse and field trials, and the biofungicide Stargus reduced disease severity caused by pathogen Phytopythium vexans on ginkgo and red maple plants in greenhouse trials. These results will help nursery producers make proper management decisions for newly reported Phytopythium crown and root rot disease of ginkgo and red maple plants.

The Fusarium solani species complex infecting cannabis (Cannabis sativa L., marijuana) plants and a first report of Fusarium (Cylindrocarpon) lichenicola causing root and crown rot

Greenhouse-grown cannabis (Cannabis sativa L., marijuana) plants with yellowing, crown rot and root-browning symptoms were sampled from six production facilities during 2019–2020. Among 34 fungal isolates recovered, 28 were identified as Fusarium solani and six isolates were provisionally identified as Cylindrocarpon sp. based on morphology. These latter isolates produced slow-growing colonies with grey-white aerial mycelium and a chestnut-brown colour below. Cylindrical 1–3 septate conidia without a distinctive foot cell were produced. Microconidia were absent and chlamydospores were produced in culture. Phylogenetic analysis of three isolates based on the elongation factor (TEF-1 α) and ITS1-5.8S-ITS2 regions identified them as Fusarium lichenicola (formerly Cylindrocarpon lichenicola), a member of F. solani species complex (FSSC) subclade 16. Pathogenicity tests using a mycelial and spore suspension were performed on cannabis cuttings and rooted plants. Isolates of F. solani originating from diseased crowns caused symptoms in 10–14 days, while those of F. lichenicola caused yellowing and wilting after 3 weeks, suggesting that F. lichenicola is less virulent. Inoculum of F. lichenicola was detected in coco coir samples used for plant propagation. Previous reports of F. lichenicola are from tropical climates, where the fungus has been associated with dermal and ocular infections of human tissues, with a few reports of it causing diseases on pomelo fruits, taro corms and tea plants. This study demonstrates the first occurrence worldwide of F. lichenicola on cannabis plants, on which it is considered a weak introduced tropical pathogen, likely to have originated from coco coir imported into Canada.

First Report of Neopestalotiopsis rosae Causing Leaf Blight and Crown Rot on Strawberry in Taiwan.

For the past 30 years, the most predominant strawberry cultivar in Taiwan has been 'Taoyuan No. 1', which produces fruit with rich flavor and aroma but is highly susceptible to anthracnose (Chung et al. 2019). Because epidemics of anthracnose became more destructive, farmers switched to an anthracnose-tolerant cultivar 'Xiang-Shui' (~50% and ~80% of the cultivation area in 2018 and 2019, respectively). Since 2018, severe leaf blight and crown rot symptoms have been observed all year in 'Xiang-Shui' in Miaoli, Nantou, Hsinchu, Taipei, Taoyuan, and Chiayi Counties. The disease became more prevalent and severe during 2019 to 2020 and caused up to 30% plant loss after transplanting. Symptoms appeared as brown necrotic lesions with black acervuli on leaves, slightly sunken dark-brown necrosis on stolons, and sunken reddish-brown necrosis on fruit. The diseased crown tissue showed marbled reddish-brown necrosis with a dark-brown margin, and plants with severe crown rot usually showed reddish-brown discoloration on leaves (the leaves initially turned reddish-brown between the veins and could become entirely scorched at later stages). To isolate the causal agent, small fragments of diseased leaves, crowns, stolons, and fruits were surface-disinfested with 0.5% sodium hypochlorite for 30 seconds, rinsed with sterile water then placed on 1.5% water agar. Single hyphal tips extended from tissues were transferred to potato dextrose agar and cultured for 7 days at 25°C under a 12-h/12-h photoperiod. Total 20 isolates were obtained from diseased leaves, crowns, stolons, and fruits. Colonies were white with cottony aerial mycelium, irregular margins, and black acervuli distributed in concentric rings. Conidia were fusiform to ellipsoid (five cells) with one basal appendage and three or four (usually three) apical appendages. From colony and conidial morphology, the causal agent was identified as Neopestalotiopsis sp. (Maharachchikumbura et al. 2014). The internal transcribed spacer (ITS) region, β-tubulin (TUB), and translation elongation factor 1-alpha (TEF-1α) of three isolates (ML1664 from diseased crown tissue collected in Hsinchu County; ML2147 and ML2411 from diseased leaves collected in Miaoli County) were sequenced (GenBank nos. MT469940 to MT469948). All three isolates clustered with the ex-type strain of Neopestalotiopsis rosae in the multilocus (ITS+TUB+TEF-1α) phylogenetic tree. To fulfill Koch's postulates, spore suspensions of ML1664 and ML2147 at 1×106 conidia/mL were used to spray-inoculate 'Xiang-Shui' seedlings at the 3 to 4 leaf stage until run-off (two trials, five seedlings per trial). Inoculated plants were put in a plastic bag (> 90% RH) at 25°C under a 12-h/12-h photoperiod. After 10-14 days, 80% of inoculated plants showed leaf or crown symptoms similar to those in the field. Control plants sprayed with sterile water showed no symptoms (4-5 seedlings per trial). The fungi were re-isolated from necrotic lesions with 100% frequency (n ≥ 3 isolates per trial), and morphological characters and ITS sequences were identical to the original ones. This is the first report of N. rosae causing leaf blight and crown rot in strawberry in Taiwan. N. rosae and N. clavispora have been reported as new threats to strawberry in several other countries (Rebollar-Alviter 2020; Gilardi 2019). Clarification of the pathogen provides a basis for developing strategies to control the emerging disease. Further studies are needed to evaluate the resistance/susceptibility of major strawberry cultivars and the fungicide sensitivity of the pathogen.

Annuals and Herbaceous Perennials Tolerant or Resistant to Phytophthora Species in the Landscape1

Abstract Plants of one or two cultivars of 16 annuals and 14 herbaceous perennials were evaluated based on desirability and anecdotal evidence of resistance to Phytophthora root or crown rot. Six plant cultivars served as susceptible controls. Three landscape beds were established in North Carolina and each was infested with three species of Phytophthora: P. nicotianae, P. drechsleri, and P. tropicalis. Plants were regularly rated for disease incidence and symptomatic plants were assayed to determine the presence of Phytophthora species. Ten cultivars of annuals and seven cultivars of herbaceous perennials did not exhibit symptoms of Phytophthora root or crown rot or other disease throughout the season (June 4 to October 15, 2018). Phytophthora spp. were recovered from seven and six cultivars of the evaluated annuals and herbaceous perennials, respectively. Phytophthora nicotianae, P. drechsleri, or P. cryptogea were recovered from a susceptible host in each landscape bed. P. tropicalis was recovered from one plant cultivar evaluated. Phytophthora cryptogea was recovered from three plant cultivars, although this species was not intentionally introduced in the landscape beds. We identified 22 plant cultivars within 13 herbaceous plant species that grew vigorously in landscape beds infested with species of Phytophthora. Index words: bedding plants, disease resistance, herbaceous perennials, landscape plants, Phytophthora nicotianae, Phytophthora drechsleri, Phytophthora tropicalis. Species used in this study: yarrow (Achillea millefolium L. ‘Desert Eve Red'), fernleaf yarrow (Achillea filipendulina Lam. ‘Moonshine Yellow'), angelonia (Angelonia angustifolia Benth. ‘ArchAngel Pink', ‘Serenita White'), annual vinca (Catharanthus roseus (L.) G. Don ‘Cora Apricot', ‘Cora Strawberry', ‘Pacifica Raspberry'), celosia (Celosia argentea L. ‘New Look'), tickseed (Coreopsis auriculata L. ‘Nana', ‘Yellow Jethro Tull'), purple coneflower (Echinacea purpurea (L.) Moench ‘Cheyenne Spirit', ‘PowWow Wild Berry'), blanket flower (Gaillardia x grandiflora Hort. ‘Goblin', ‘Mesa Bi-color'), Barberton daisy (Gerbera jamesonii Bolus ex Hooker f. ‘Crazy Daisy'), verbena (Glandularia canadensis ‘Homestead Purple'), >dusty miller (Jacobaea maritima (L.) Pelser & Meijden ‘Silver Dust'), New Guinea impatiens (Impatienshawkeri W.Bull ‘Hamony', ‘Sunpatiens Compact Orchid', ‘Sunpatiens Lilac'), sweet potato vine (Ipomoea batatas (L.) Lam. ‘Ace of Spades', ‘Bright Idea Tri-color'), West Indian lantana (Lantana camara L. ‘Miss Huff'), lantana (Lantana x hybrida ‘New Gold'), shasta daisy (Leucanthemum x superbum (Bergmans ex J.W. Ingram) Bergmans ex Kent. ‘Becky', ‘Snow Lady'), bee balm (Monarda didyma L. ‘Petite Delight', ‘Jacob Cline'), ornamental grass (Panicum virgatum L. ‘Rotstrahlbusch', ‘Shenandoah'), geranium (Pelargonium x hortorum L.H. Bailey (pro. sp.) ‘Bullseye Cherry', Calliope Dark Red'), calibrachoa (Petunia x calibrachoa ‘Super Cal'), petunia (Petunia x hybrida (Hooker) Vilmorin ‘Easy Wave Red', ‘Easy Wave White', ‘Wave Purple', ‘Yellow Madness', Violet Picotee'), annual phlox (Phlox drummondii Hook. ‘Intensia Red Hot', ‘Phlox Star'), garden phlox (Phlox paniculata L. ‘Amethyst True Gal'), black-eyed susan (Rudbeckia hirta L. ‘Indian Summer', ‘Prairie Sun'), mealy blue sage (Salvia farinacea Benth. ‘Victoria Blue'), African marigold (Tagetes erecta L. ‘Inca Yellow', ‘Proud Yellow'), French marigold (Tagetes patula L. ‘Disco Mix', ‘Disco Yellow'), narrowleaf zinnia (Zinnia angustifolia Kunth. ‘Star Orange', ‘Star White'), Phytophthora nicotianae Breda de Haan, Phytophthora cryptogea Pethybr. and Laff, Phytophthora drechsleri Tucker, Phytophthora tropicalis Aragaki and J.Y. Uchida, zinnia (Zinnia elegans Jacq. ‘Magellan Orange').

Cylindrocladiella peruviana and Pleiocarpon algeriense causing stem and crown rot on avocado (Persea americana)

During the winter of 2018, 3-years-old trees of avocado (Persea americana) cv “Hass” from Trapani province (Sicily, Italy) showed symptoms of stem and crown rot. Two different fungi were consistently isolated from infected tissues. Morphological characterization and multi-locus phylogenies using five genomic loci (ITS, tef1, tub2, his3, and rpb2) identified these fungi as Cylindrocladiella peruviana and Pleiocarpon algeriense. Pathogenicity tests on healthy 5-months-old seedlings and 3-year-old trees of avocado reproduced similar symptoms as those observed in nature, and Koch’s postulates were fulfilled for both pathogens. Moreover, the tested fungal isolates revealed a different pathogenic behaviour among two species. Two isolates of Pleiocarpon algeriense resulted more aggressive than Cylindrocladiella peruviana isolates causing major lesions on young seedlings. This study is the first to report of stem and crown rot on avocado caused by Cylindrocladiella peruviana and Pleiocarpon algeriense.

First detection of Fusarium oxysporum f. sp. radicis–lycopersici across major tomato–producing regions in Brazil

Fusarium oxysporum f. sp. lycopersici (FOL) races 1 and 2 were, up to recent years, the only pathogenic Fusaria associated with tomatoes (Solanum lycopersicum L.) in Brazil. However, recent outbreaks of F. oxysporum isolates with the ability to breakdown the I–2-mediated resistance prompted us to conduct nationwide surveys of the pathogens associated with vascular wilt of tomatoes. During these surveys, eight atypical F. oxysporum isolates (collected across five tomato–producing Brazilian states) displayed a peculiar ability of infecting accessions carrying the I–3 and I–7 genes (controlling resistance to FOL race 3 isolates). These observations suggested the involvement of either a new FOL race or of F. oxysporum f. sp. radicis-lycopersici (FORL) isolates. Koch’s postulates were fulfilled for these eight atypical isolates via root dipping inoculation (106 conidia mL− 1) of seedlings from two tomato cultivars ‘Ponderosa’ and ‘Dominador’. All eight isolates induced crown-rot symptoms only in ‘Ponderosa’, but not in ‘Dominador’ (which is resistant to FORL). To confirm the FORL identity, PCR assays were done using a set of race-specific and forma specialis-specific primers. A single amplicon of ≈ 947 bp was observed with the primer pair Sprl only with DNA template obtained from standard FORL isolates and from the eight atypical isolates, confirming the first detection of this forma specialis in Brazil. The geographical distribution of FORL isolates across major tomato–producing areas may require the incorporation of resistant factors into adapted cultivars.

First Report of Phytopythium helicoides Causing Root and Crown Rot on Strawberry in China

Strawberry (Fragaria × ananassa Duch.) is an important cash crop in China. According to the 2016 China Agricultural Yearbook, Jiangsu Province is an important player in China’s strawberry market, with a cultivation area of 18,100 ha and production of 460,000 metric tons per year. We investigated a commercial field in the Jiangsu Province where the strawberry cultivar HongYan had been cultivated for more than 5 years. Between September and December 2017, symptoms of root and crown rot appeared in strawberry plants: plants developed disease symptoms characterized by yellowing of lower leaves, wilt, blight, and often death. The roots had a dark brown rot, and crowns were also rotted. The average incidence of the disease was up to 25%, and it even led to destruction and closure of a certain strawberry greenhouse. Root tissues (5 mm²) from 25 infected plants were excised from the central rotted root, surfaced sterilized by soaking in 75% ethyl alcohol for 30 s, rinsed three times in sterilized distilled water, and plated onto potato dextrose agar (PDA). All 25 isolates from each infected plant had the same morphological characteristics. They were able to grow at temperatures between 15 and 40°C, and there were no distinctive colony growth patterns on PDA. The sporangia were obovate or subglobose, papillate, and 21 to 34 × 20 to 28 µm in diameter. Oogonia were smooth and 22 to 30 µm in diameter. To confirm the identity of the pathogen, the rDNA internal transcribed spacer region (ITS, NCBI accession no. MT179561), cytochrome c oxidase I gene (cox I, MT188654), and cytochrome c oxidase II gene (cox II, MT188655) of three isolates were amplified and sequenced using the primer pairs ITS1/ITS4 (White et al. 1990), FM35/FM59, and FM66/FM58 (Martin 2000), respectively. BLAST analysis showed in all cases 98 to 100% identity with segments of Phytopythium helicoides (GenBank accession nos. MG597190.1 for ITS, KT258906.1 for cox I, and KT258907.1 for cox II). Based on the morphological features and the phylogenetic analyses of DNA sequences, the isolates were identified as P. helicoides. Pathogenicity assays were conducted on 3-week-old HongYan strawberry seedlings in September 2018. The roots of each seedling were dipped for 10 min into zoospore suspension of each isolate (1 × 10⁶ zoospores/ml), and the control seedlings were inoculated using the same procedure with sterilized distilled water. Five replicate seedlings were inoculated and planted individually into 1.8-liter free-draining pots containing sterilized soil. All the seedlings were incubated at 22°C under a 16-h light/dark regime and watered with 50 ml every 5 days. All inoculated plants showed root necrosis and crown rot 20 days after inoculation. The symptoms of inoculated seedlings were similar to the naturally infected plants. The same pathogen was reisolated from symptomatic roots and crowns. No symptoms were observed in the control seedlings. Although P. helicoides has been reported causing root and crown rot on strawberry in Japan and America (Ishiguro et al. 2014; Marin et al. 2019), rhizome rot on Asian lotus, and stem rot on Shatangju mandarin in China (Chen et al. 2016; Yin et al. 2016), it has not been reported in China from strawberry. Thus, strawberry growers should consider this pathogen when diagnosing and managing plant diseases in this crop.

A phylogenomic tree of fungi: evolutionary relationships among Calonectria ilicicola and 586 fungal mitochondrial genomes

The mitochondrial genomes have the potential to be ‘molecular clock’ due to its high mutation rate and low DNA recombination rate. Here, we sequenced the complete mitochondrial genome of three strains of Calonectria ilicicola (anamorph Cylindrocladium parasiticum) isolated from diseased peanut showing symptoms of Cylindrocladium black rot (CBR) and two C. ilicicola strains isolated from diseased soybean showing symptoms of Red crown rot (RCR). The complete mitogenome of C. ilicicola ranged from 41,187 bp to 64,421 bp in length, 22–34 protein-coding genes, 19–20 tRNA genes, 2 rRNA genes, and a control region. Besides, we used a phylogenomic approach to infer evolutionary relationships among 591 mitochondrial genomes that span the diversity of 294 fungal species, including C. ilicicola. Our results underline the potential importance of mitochondrial genomes in comparative genomic analyses and provide a robust evolutionary insight across the tree of life.

EFFCIENCY OF SOME BIOLOGICAL AND CHEMICAL TREATMENTS AGAINST WHEAT ROOT AND CROWN ROT DISEASE

Fusarium culmorum(W.G. Smith) Sacc., Bipolaris sorokiniana (Sacc.) Shoemaker, Rhizoctonia oryzae Kuhn and Fusarium spp. were isolated from wheat plants exhibiting typical root and crown rot symptoms from different districts at Sharkia Governorate, Egypt during 2015/ 2016 growing season. Pathogenicity test revealed that B. sorokiniana was the most virulent one causing pre-emergence damping-off followed by Fusarium culmorum. In addition, F. culmorum was the most virulent one responsible for post-emergence damping-off incidence. Rhizoctonia oryzae showed the highest percentage of root rot, Moreover, F. culmorum and B. sorokiniana showed the highest percentage of disease incidence. In the same trend, F. culmorum induce the highest percentage of disease severity. In vitro, Trichoderma sp. bio-agent and its culture filtrate were the most effective treatment that reduced mycelial growth of the tested fungi. In vivo, it decreased pre and post-emergence damping off, root rot, disease incidence and disease severity compared with the control. In addition, the obtained results indicated a significant increase on healthy survival plants and significantly improved the plant growth parameters i.e. fresh and dry weights of shoots and roots, plant height, spike length and 1000 grain weight. In vitro,Score and Amistar-top fungicides were the most effective in inhibiting the mycelial growth of R. oryzae followed by Amistar-top on B. sorokinana and Score onF. culmorum.Score was the most effective treatment revealed the highest percentage of healthy survival plants followed by Amistar-top and gave the highest protection against root and crown rot disease as shown by disease incidence and severity percentages.

Identification and pathogenicity of Fusarium spp., the causal agent of wheat crown and root rot in Iran

Crown and root rot are among the most important wheat diseases caused by Fusarium species. The aim of this study was the identification of Fusarium spp. isolates obtained from wheat with crown and root rot symptoms in Yazd province of Iran, based on morphological and molecular characteristics. The ef1/ef2 and ITS4/ITS5 primers were used for molecular identification of the isolates. The results of morphological and molecular investigations revealed that among 40 isolates, 11 isolates were identified as F. equiseti, six isolates as F. pseudograminearum, five isolates as F. culmorum, five isolates as F. flocciferum, four isolates as F. acuminatum, four isolates as F. oxysporum, three isolates as F. solani and two isolates as F. proliferatum. Pathogenicity of the isolates was evaluated on wheat cultivar Falat. Comparing pathogenicity of different Fusarium spp. isolates on wheat seedlings revealed that the highest level of disease index was observed for F. pseudograminearum, F. culmorum, F. flocciferum and F. solani and the lowest disease severity was observed for F. equiseti. This is the first report on the identification of Fusarium spp., causing wheat crown and root rot in Yazd province of Iran, and on pathogenicity of F. flocciferum on wheat.

Fusarium Crown Rot Whitehead Symptom as Influenced by Wheat Crop Management and Sampling Date.

Symptoms of Fusarium crown rot of wheat include premature death of inflorescens (whiteheads), lesions on subcrown internodes, and rotting of crown tissue and lower stem internodes. Each symptom type is influenced by a different set of environmental conditions. Whiteheads are the easiest symptom to quantify and are frequently reported in the Pacific Northwest U.S.A. The objective of this research was to examine factors associated with whitehead expression and relationships with wheat yield and test weight. Incidence of whiteheads differed for inoculations with different isolates of F. pseudograminearum and F. culmorum, and over years due to weather factors. Whiteheads became less as planting dates for winter wheat were delayed until after September, and incidence was increased with increasing nitrogen application rate. Dates of initial and greatest expression of whiteheads differed among cultivars, which was associated in part with the cultivar heading date. Whiteheads were not correlated with subcrown internode lesions or browning of crown tissue. Whiteheads were also not correlated with grain test weight. Whiteheads were sometimes negatively associated with grain yield, but that relationship was variable and could not be considered a reliable, recurrent, or accurate measure of crown rot severity. These results indicate the need for caution in reporting whiteheads as a sole indicator of cultivar susceptibility to Fusarium crown rot.

First Report of Anthracnose Crown Rot of Strawberry Caused by Colletotrichum siamense in Taiwan

In Taiwan, strawberry (Fragaria × ananassa Duch.) is a high-value crop with an average annual cultivated area of ∼500 ha in the last 5 years. Over 90% of strawberry cultivation is in Miaoli County, with ‘Taoyuan No. 1’ as the predominant cultivar for more than 30 years. Anthracnose has become more destructive over the past decade. Although Colletotrichum gloeosporioides, C. dematium, C. fragariae, and C. acutatum were mentioned as the causal agents of strawberry anthracnose in Taiwan (Plant Protection Information System; https://otserv2.tactri.gov.tw/ppm/), we lack information on the isolation, pathogenicity, and morphological or molecular identification of the pathogen. From 2010 to 2016, we surveyed anthracnose in strawberries in Miaoli County; more than 50% of diseased plants showed typical anthracnose crown rot (ACR) symptoms (McInnes et al. 1992). ACR caused up to 30 to 40% plant loss during the seedling stage and ∼20% after transplanting. Infected crown tissue initially showed red and white marbling and then gradually brown rot, followed by rapid wilting of the entire plant. Anthracnose symptoms were observed in other parts of the plant, including leaves, petioles, runners, fruits, and roots. Symptoms appeared as circular black spots on the leaves and withering and girdling on runners. To isolate the causal agent, approximately 0.2 × 0.2-cm fragments of diseased crowns were surface disinfested with 1% sodium hypochlorite, triple rinsed with sterile water, and then placed onto 1.5% water agar. After 2 to 3 days, extended single hyphal tips from tissues were transferred to potato dextrose agar and incubated for 7 days at 25°C under a 12-h/12-h photoperiod. Colonies were initially white, later became somewhat zonate, velvety, light gray on the upper side and gray on the reverse side of plates, with concentric rings of salmon sporodochia. Conidia were 9.68 to 17.95 × 3.88 to 5.84 µm (14.53 ± 0.31 × 4.99 ± 0.08 µm, n = 90), hyaline, oblong to cylindrical, with round obtuse ends. Morphological characteristics of the causal agent resembled species belonging to the C. gloeosporioides species complex (Weir et al. 2012). To confirm the species identification, we extracted genomic DNA from 10 isolates by using the Plant Genomic DNA Extraction Miniprep System (Viogene, Taipei) and polymerase chain reaction–amplified the internal transcribed spacer (ITS) region, chitin synthase (CHS-1), actin (ACT), β-tubulin 2 (TUB2), calmodulin (CAL), and intergenic region of Apn2 and MAT1-2-1 (ApMAT) with published primers (Carbone and Kohn 1999; O’Donnell and Cigelnik 1997; Silva et al. 2012; Weir et al. 2012; White et al. 1990). Sequences were submitted to GenBank (accession nos. MK174223 [ITS], MK174224 [CHS-1], MK174225 [ACT], MK174226 [TUB2], MK174227 [CAL], and MK174228 [ApMAT]). The ITS, CHS-1, ACT, TUB2, CAL, and ApMAT sequences were compared with the GenBank nr database, restricted to type material. Results showed 98 to 99% identity to C. siamense (syn. C. dianesei and C. melanocaulon) (Liu et al. 2016; Prihastuti et al. 2009), which belongs to the C. gloeosporioides species complex, with the corresponding sequences (ITS: NR_144800; CHS-1: KX094094; ApMAT: KX094304 [Lima et al. 2013]; ACT: KX093987; TUB2: KX094290; and CAL: KX094036 [Doyle et al. 2013]). Koch’s postulates were fulfilled for two isolates (ML133 and ML612) by spraying 1 × 10⁶ conidia/ml suspension on seedlings until run-off at the four- to five-leaf stage (two trials per isolate, n = 5 seedlings per trial). Inoculated plants were covered with plastic bags (>90% relative humidity) for 24 h at 30°C and then maintained in a growth chamber at 30°C, 70% relative humidity, under a 12-h/12-h photoperiod. After 7 days, all inoculated plants showed typical necrotic leaf spots and wilt symptoms similar to those in the field. Control plants sprayed with sterile water had no symptoms (n = 5 per trial). Longitudinal sections of the inoculated crown showed reddish-brown and white-marbled necrosis. The fungi were reisolated from lesions of diseased leaves or crowns with 100% frequency (n ≥ 3 isolates per trial), and morphological characteristics and gene sequences were identical to the original isolates. To our knowledge, this is the first report of C. siamense causing ACR of strawberry in Taiwan. The disease has the potential for causing serious losses to the strawberry industry in Taiwan, and research is needed on management strategies to minimize losses.

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.