FirstReportofPythium...

T. E. Sigobodhla,S. Dimbi, andA. J. Masuka

Abstract

Pythium species have a wide host range and are important pathogens of many agricultural crops. In Zimbabwe, 15 isolates of Pythium have been obtained from symptomatic tobacco (Nicotiana tabacum) in the new float seedling production system. This production system now accounts for 25 to 30% of the tobacco industry's annual requirement of 975 million seedlings. Disease symptoms are observed usually 5 to 6 weeks after sowing as wilting and yellowing of leaves followed by rotting of the roots, and in severe cases, seedling death. Up to 70% seedling loss has been reported in commercial seedbeds. In a study to fulfill Koch's postulates and to determine the susceptibility of 16 commercially grown tobacco cultivars, seedlings were produced in float trays and inoculated individually with 1 ml of Pythium (Isolate Py 19) spores (1 × 10⁴ CFU/ml) and mycelium pipetted around the base of the stem of each seedling 9 weeks after sowing. First symptoms appeared 7 to 10 days after inoculation as yellowing and wilting of leaves. When seedlings were pulled, the lower portion of the stem and roots were brown and rotted. Seedling mortality averaged 29% and disease incidence was 96 to 100% among cultivars. All 16 tobacco cultivars were susceptible to Pythium root and stem rot and there were no significant (P > 0.05) differences in their susceptibility to the disease. The pathogen was reisolated from the inoculated seedlings. The representative isolate (Py 19) sent to Centraalbureau voor Schimmelcultures, the Netherlands was identified as Pythium myriotylum Drechsler (CBS Accession No. 125021) (2). P. aphanidermatum, causing black stem rot, and P. debaryanum and P. ultimum, responsible for damping-off (3), have been reported in the predominant conventional soil-based tobacco seedling production system, but do not cause economic losses. However, stem and root rot caused by P. myriotylum threaten the float seedling production system in Zimbabwe, although a chemical curative control of the disease has been recommended and is now widely practiced. P. myriotylum has previously been reported in the tobacco float seedling production system in South Carolina (1). To our knowledge, this constitutes the first published report of P. myriotylum on tobacco in Zimbabwe.

References: (1) M. G. Anderson et al. Plant Dis. 81:227, 1997. (2) Centraalbureau voor Schimmelcultures. Retrieved from http://www.cbs.knaw.nl, 2010. (3) A. J. Masuka et al. List of Plant Diseases in Zimbabwe. Department of Research and Extension and Tobacco Research Board. Harare, Zimbabwe, 2003.

First report of root rot caused by Pythium aphanidermatum and pythium -group on hydroponically grown pepper in Bulgaria

In September 2013 and July 2014, severe wilt symptoms were observed on mature, sweet pepper (Capsicum annum L.) plants (c. Sofijska kapiya) grown in a rockwool hydroponic system in a large-scale commercial greenhouse near Petrich, South West of Bulgaria. Examination of the wilted plants revealed progressive chlorosis of the lower leaves, severe brown and soft root rot and basal stem rot. Less affected plants showed yellow discoloration of the root system, stunted growth and lack of vigor. Approximately 25% of the pepper plants collapsed and died particularly during the fruiting period in the first year of observations and more than 30% of plants died in the second year. Isolations were made from sections of roots and basal stems of pepper plants with symptoms of disease on standard, nonselective media such as oatmeal (OA), potato dextrose (PDA) or water (WA) agar media. Plates were incubated at 25°C in the dark for 7 days. Pythium spp. isolates were readily obtained from all pepper plants with disease symptoms as well as from symptomless plant roots. © 2016, National Centre for Agrarian Sciences. All rights reserved.

First report of Pythium aphanidermatum causing rootrot on common ice plant (Mesembryanthemumcrystallinum)

Common ice plant (Mesembryanthemum crystallinum) is a facultativehalophyte, originating in South African deserts (Huxley et al., 1992), whichhas recently been cultivated for edible use in Japan. In June 2012, a severeroot rot was found on commercially grown ice plants in a greenhouse inOsaka, Japan. Root rot appeared suddenly on five- to six-week-old plants(Fig. 1a), and approximately 300 plants in the greenhouse (i.e. a quarter ofall the ice plants grown) were found to be affected by the disease. Theplants were grown in a conventional hydroponic system that used 'OtsukaA' nutrient solution (OAT Agrio Co., Ltd. Tokyo, Japan), amended with0.1% sea salt. The greenhouse had a controlled temperature regime(23/18°C, day/night), and 16 hours of artificial light per day. The affectedtissues were soft and discoloured, and wilting of the plants was observed(Fig. 1b). Abundant aplerotic oospores were found in diseased roots (Fig.1c). A Pythium-like organism was isolated and identified as P.aphanidermatum based on morphological characters and hyphal growth rateat different temperatures. The observed morphological characters were asfollows: main hyphae up to 10 μm wide; sporangia mostly terminal,sometimes intercalary and consisting of inflated structures (Fig. 1d);oogonia terminal, globose, smooth, 21.0–26.9 μm in diameter; antheridiaintercalary, sometimes terminal, 10.4–15.5 μm long and 8.1–11.5 μm wide,one per oogonium; oospores aplerotic, 14.2–22.8 μm in diameter, oosporewall 1.0–2.0 μm thick (Fig. 1e); and zoospores formed at 25-30°C.Cardinal temperatures for growth on potato carrot agar were 10°Cminimum, 37°C optimum, and 40°C maximum, with a daily radial growthrate of 30.8 mm at 25°C. The ITS region of the representative isolateOPU852 was amplified and sequenced with primers ITS4 and ITS5 (Whiteet al., 1990). Sequence analysis determined 100% identity to P.aphanidermatum isolate CBS118.80 (GenBank Accession No. HQ665084;Robideau et al., 2011). The sequence generated in this study was depositedin GenBank (KT336808) and isolate OPU852 was deposited in the NIASGenebank, Ibaraki Prefecture, Japan as isolate no. MAFF245234. A pathogenicity test was conducted using isolate OPU852 in a small-scalehydroponic system that had the same nutrient solution, and temperature andlight conditions as described above. A total of 30 thirty-day-old ice plantswere transplanted into the system. Pythium aphanidematum zoospores werereleased and prepared as described by Raftoyannis & Dick (2002), and 90ml of pond water containing approximately 104 zoospores/ml was pouredinto the hydroponic system. After seven days incubation, wilting symptomswere observed on 50% of the inoculated plants, whereas no evidence ofdisease was observed in a non-infected hydroponic system. The pathogenwas re-isolated from diseased plant roots and confirmed as P.aphanidermatum. In Japan, Pythium aphanidermatum is a devastating pathogen on manyplants, especially on common bean and sugar beet, and has been reportedsince 1935 (van der Plaats-Niterink, 1981). However, it has never beenreported on common ice plant. To our knowledge, this is the first report ofP. aphanidermatum causing root rot on common ice plant worldwide.Moreover, no Pythium spp. have been recorded previously from this plant.Hydroponic systems might favour the development of this disease due tothe environmental conditions that promote the growth of the pathogen(Stanghellini & Rasmussen, 1994).

First Report of Pythium aphanidermatum Crown and Root Rot of Industrial Hemp in the United States

(February 2017 Plant Disease ; 101(6)DOI:10.1094/PDIS-09-16-1249-PDN)

During June and July 2015, crown and root rot symptoms were observed on industrial hemp (Cannabis sativa cvs. Alyssa and Canda) in research plots in Lafayette, IN. Record setting rainfall in Indiana during June (218.4 mm) and July (162.6 mm) may have factored into this outbreak. Soil type is Crosby-Miami complex alfisol. Symptom development appeared 13 days after sowing with temperatures ranging from 25 to 30°C. Leaves of affected plants were chlorotic, and plants were stunted and often wilted. Brown lesions on roots and loss of feeder roots were observed when symptomatic plants were removed from soil; symptomatic plants often but not always possessed brown, water-soaked stem lesions. A small percentage of affected plants collapsed, but most persisted in stunted growth. Thin, aerial mycelia were visible on the stem surface of some of the infected plants. Tissue fragments were excised from the margins of the affected stem and root lesions, surface sterilized, and plated on 0.25% potato dextrose agar (PDA) and on a medium selective for oomycetes containing pimaricin, ampicillin, rifampicin, and pentachloronitrobenzene (PARP). Plates were incubated at 22°C in the dark. Thirty-eight isolates were tentatively identified as Pythium spp. and were grown on V8 medium (V8 juice 300 g; agar 15 g; CaCO3 1.5 g; distilled water 1 liter) for morphological observation. DNA was extracted with the G-Biosciences OmniPrep for fungi. PCR was performed using the primers ITS1/ITS4 to amplify the internal transcribed spacer (ITS) region of rDNA. Six isolates were identified as Pythium aphanidermatum by BLAST analysis (Altschul et al. 1997) and were morphologically consistent with P. aphanidermatum (Watanabe, 2002). One characteristic isolate with a 738-bp segment showed a 99% homology with the sequence of P. aphanidermatum (GenBank accession JN695786) and has been assigned as GenBank accession KX772239. Pathogenicity tests were performed with this isolate using 20 seeds of 'Canda’ sown in three 1-liter trays filled with a steam-disinfested soilless medium (Sungro Professional Growing Mix). Plants were fertilized 1 day before inoculation with 0.9 g ammoniacal nitrogen. Plant were inoculated 21 days after sowing (postemergent) with sterilized hemp kernels or 0.25% PDA colonized with the one isolate of P. aphanidermatum. Infested and control inoculum was placed in rows 5 mm from plants. One-liter trays were placed in two-liter trays and watered to flooding to simulate waterlogged conditions. Greenhouse temperature was 25°C; 12 h day night light cycle supplementing and external light. This experiment was replicated three times for both control and challenged treatments. Symptoms developed 18 days after inoculation. After 31 days, all inoculated plants displayed symptoms of chlorosis, crown rot, wilt, dieback, and eventually death. Control plants remained healthy throughout the experiment despite waterlogged conditions. This experiment was repeated 3 days later, with symptoms first developing 14 dpi. P. aphanidermatum was consistently observed based upon morphological observation and/or reisolation from the lesions. To our knowledge, this is the first report of Pythium root and crown rot of C. sativa caused by P. aphanidermatum in the U.S. Although current production of industrial hemp is only 6,900 acres, the importance of the disease could increase with increasing hemp production when it is legalized, particularly in low lying or flood prone areas where hemp is intensively grown (McPartland 1996). © 2017, American Phytopathological Society. All rights reserved.

First Report of Anthracnose on Cynanchum atratum Caused by Colletotrichum destructivum in China

Cynanchum atratum Bunge is a perennial herb used as a medicinal plant and occasionally as an ornamental plant in China. In September 2014, light brown spots (3 to 9 mm) with nut-brown annulations that gradually enlarged and turned black in the center were observed on leaves of C. atratum in Changchun Medicinal Garden (125.24°E, 43.48°N), Jilin Province, China. The disease symptoms on C. atratum leaves were believed as anthracnose. To identify the causal agent, small pieces of symptomatic leaves were surface sterilized with 75% ethanol for 1 min, followed by 0.1% NaOCl for 1 min, washed with sterile distilled water three times, transferred to PDA media, and kept at 25°C under a 12-h light/dark photoperiod. A fungus was consistently grown from the symptomatic tissues. Five single conidial isolates were obtained and transferred to fresh PDA plates for 7 days. Colony and conidia morphology of five isolates were observed. The colonies were white to light brown. Hyphae were branched and hyaline. The size of conidia was 17.0 (13.9 to 20.2) × 5.6 (3.8 to 7.4) μm, and appeared as hyaline, oblong, straight or slightly curved, and sometimes with two oil globules. Acervuli were dark brown to black. Brown and straight or slightly curved setae (30.8 to 94.6 μm long, avg. 54.6 μm) were formed on acervuli. Salmon-colored mucilaginous masses were also produced on PDA. Based on the morphological characteristics, isolates were preliminarily identified as Colletotrichum destructivum (O’Gara) (Damm et al. 2014). The identification as C. destructivum was confirmed by amplifying and sequencing of the ITS region of rDNA (ITS5/ITS4) (Damm et al. 2014; White et al. 1990), partial actin gene (ACT-512F/ACT-783R), chitin synthase1 gene (CHS-79F/CHS-345R) (Carbone and Kohn 1999), and glyceraldehyde-3-phosphate dehydrogenase gene (GDF1/GDR1) (Guerber et al. 2003) for each of the five isolates. The results showed that these five isolates were identical and the sequences of the ITS, ACT, CHS1, and GAPDH genes of isolate BW-1 were deposited in GenBank. The sequence of ITS regions (LT174650) showed 98.37 and 98.19% identity with sequences of C. destructivum CD-hz 01 (EU070911) and MAFF 239948(AB334522), respectively. The sequence of CHS1 gene (KU878966) showed 99.59 and 99.18% identity with sequences of CBS149.34 (JQ005785) and CBS136228 (KM105227). The sequences of ACT gene (LT174651) showed 98.71 and 98.28% identity with sequence of C. destructivum CGMCC3.15128 (KC843544) and CY-1 (KP262347). The sequence of GAPDH gene (KX431142) showed 97.44% identity with C. destructivum CBS136228 (KM105561) (Damm et al. 2014). To complete Koch’s postulates, 10 healthy plants for each isolate were spray-inoculated with a conidial suspension (1 × 10⁵ conidia ml^–1) and 10 control plants were spray-inoculated with sterile distilled water. All plants were maintained at 25°C and 90% RH with a 12-h photoperiod. Anthracnose symptoms that resembled those in naturally developed C. atratum leaves were observed 5 days after inoculation. No symptoms were observed on control plants. Pathogenicity tests were repeated twice. C. destructivum was obtained from the inoculated plants and the reisolates were the same as that used for inoculation. Currently, the knowledge regarding economic importance of anthracnose on C. atratum is limited; however, with increasing cultivation area of C. atratum, anthracnose is likely detrimental to economics in C. atratum production. To our knowledge, this is the first report of anthracnose on C. atratum Bunge caused by C. destructivum in China.