First report of strains within the Pythium spinosum species complex causing carrot cavity spot in California

Carrots (Daucus carota L. subsp. sativus (Hoffm.)) with typical symptoms of cavity spot, i.e., sunken, round to elliptical lesions of 2-5 mm long (Hiltunen and White 2002), were collected from two locations in California, Bakersfield and Riverside, in January and July 2019, respectively. Carrots were rinsed in tap water, 4-mm2 lesion fragments were pressed into selective corn meal agar (CMA-PARP; Schrandt et al. 1994) and incubated at 23ºC in the dark for four days. Identification of pure cultures was performed via amplification and sequence analysis of two genomic regions, the Internal Transcribed Spacer 1-5.8S-ITS2 (ITS) region and the cytochrome C oxidase subunit 1 (COI) gene, using the universal primers UN-UP18S42/UN-LO28S576B (Schroeder et al. 2006) and OomCOXI-Levup/OomCOXI-Levlo (Robideau et al. 2011), respectively. Via BLAST, two isolates from organically grown carrots in Bakersfield (MCIF19) and Riverside (JSCS19), with identical ITS sequences (GenBank Acc. Nos. MZ799354 and MZ799355, respectively), showed 99.61% similarity (1021/1025 bp) to that of Pythium spinosum (AY598701.2). Yet, the COI of MCIF19 (MZ803207) showed 98.72% similarity (692/701 bp) to that of Pythium paroecandrum (GU071818.1), while the COI of JSCS19 (MZ803208) was identical (701/701 bp) to that of Pythium kunmingense (GU071820.1), a rarely isolated species considered within the species complex of P. spinosum (Robideau et al. 2011). According to these results, the isolates were identified as belonging to the P. spinosum species complex, part of Pythium Clade F (Lévesque and De Cock 2004; Robideau et al. 2011). Further research is needed to clarify the exact taxonomic status of both isolates. Koch’s postulates were completed using two different assays. Each assay was done twice and with carrots of the cultivar Maverick. Surface-sterilized, freshly harvested, mature carrots, in a plastic box lined with moistened sterile paper towels, were inoculated each with four CMA plugs (5-mm diameter) with actively growing mycelium of each isolate. CMA plugs, non-inoculated or colonized by a known pathogenic P. violae strain, were used as the negative and positive control, respectively. Boxes were closed to maintain humidity and incubated at 23ºC in the dark. Lesions similar to the ones caused by P. violae were observed at day 3 for all plugs of both isolates. No symptoms were observed for the negative control, even after extending the incubation to 7 days. In a more natural assay, four non-treated carrot seeds were planted in tree seedling pots (25 x 6.5 cm) containing sterilized 50/50 peat moss/sand combined with 15-ml V8 broth (Schrandt et al. 1994) with densely grown mycelium. The same inoculation treatments were used as for the carrot disk assay. Plants (one plant/pot, four plants/treatment) were maintained at 23ºC under a 16 h photoperiod with daily watering (20 ml). At 14 weeks, the carrots inoculated with P. violae and the two test isolates showed cavity spot lesions while no symptoms were observed on carrots growing in non-inoculated medium. For both assays, pathogens were re-isolated from rinsed symptomatic tissue and their identity was confirmed using the molecular analysis described above. No oomycetes were recovered from the non-inoculated carrots. Although several Pythium species have been associated with cavity spot before, this is, to our knowledge, the first report of strains within the P. spinosum species complex causing carrot cavity spot in California and elsewhere. Funding: This research was made possible by the California Fresh Carrot Advisory Board (FRA-21). References: Hiltunen, L.H., and White, J. G. 2002. Ann. Appl. Biol. 141:201. Lévesque, C. A., and De Cock, W. A. M. 2004. Mycol. Res. 108:1363. Robideau, G.P., et al. 2011. Mol. Ecol. Resour. 11:1002. Schrandt K. K., et al. 1994. Plant Dis. 78:335. Schroeder, K.L., et al. 2006. Phytopathology 96:637. Supplementary material: Supplementary figure S1 Supplementary figure S2

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First Report of Fusarium oxysporum and F. solani Causing Fusarium Dry Rot of Carrot in China

Plant Disease ◽

10.1094/pdis-02-14-0156-pdn ◽

2014 ◽

Vol 98 (9) ◽

pp. 1273-1273 ◽

Cited By ~ 1

Author(s):

X. Y. Zhang ◽

J. Hu ◽

H. Y. Zhou ◽

J. J. Hao ◽

Y. F. Xue ◽

...

Keyword(s):

Plant Protection ◽

Tap Water ◽

Disease Incidence ◽

Blue Pigment ◽

Universal Primers ◽

Infested Soil ◽

Single Spore ◽

Dry Rot ◽

First Report ◽

Daucus Carota L

Carrot (Daucus carota L.) is an economically important vegetable crop in China. In August 2008, a disease was observed on carrot in Inner Mongolia. The symptoms appeared as dry rot lesions on root surface, expressing light brown cankers with defined rounded or irregular shapes (1,3). The average disease incidence was up to 80% in Tuo Ke Tuo County. The disease has been a serious problem in these two counties since then, especially where consecutive carrot cropping was practiced. Carrot roots with typical dry rot symptoms were washed with tap water. Root tissues near the margin of necrotic lesions were excised, surface sterilized with 1% NaOCl for 3 min, and rinsed with sterile distilled water three times. The disinfected tissue was placed on potato dextrose agar (PDA) in a petri dish. Plates were incubated at 25 ± 1°C in the dark for 4 days. Fusarium single spore isolates were obtained from characteristic colonies (1). Three isolates (CF1, CF2, and CF3) were used for further study. The isolates were identified as Fusarium spp. on the basis of microscopic morphology on PDA. CF1 produced pink pigment, abundant falciform macroconidia of 14.7 to 38.2 × 4.5 to 5.7 μm with 2 to 3 septates, and elliptic microconidia of 7.5 to 15.1 × 3.3 to 5.4 μm with none or one septate. CF2 and CF3 produced light blue pigment, abundant falciform macroconidia of 16.4 to 34.4 × 4.0 to 6.1 μm with 2 to 3 septates, and elliptic microconidia of 6.7 to 10.7 × 3.0 to 4.9 μm with none or one septate. They were further identified and confirmed by PCR. The PCR involved amplifying the internal transcribed spacer (ITS) region of ribosomal DNA using genomic DNA as the template with universal primers ITS1 and ITS4 (2). The PCR products were sequenced. BLAST analysis of these sequences against the GenBank database determined the taxonomy of the isolates. The sequence of CF1 was 99% identical to F. oxysporum (Accession No. KC594035); sequences of CF2 and CF3 were 99% identical to F. solani (KC215123). To confirm the pathogenicity of the isolates, mature carrot roots (cv. Hong Ying 2) were inoculated with mycelial plugs (5 mm in diameter) cut from the margin of actively growing colonies on PDA plates. One mycelial plug was placed on each carrot root, with the mycelial side facing the root. PDA plugs were used for controls. Each treatment had five replicates. The inoculated roots were incubated in a humid chamber (90% RH) at 25°C. Four days after incubation, mycelia of the isolates developed and covered most of the surface of carrot roots, and brown rot lesions were observed on all inoculated roots, while the controls remained symptomless. This experiment was repeated. In another trial, carrot seeds (cv. Hong Ying 2) were sown in sterilized soil in pots (30 × 25 cm opening) with 15 seeds per pot. The soil was infested with either CF1, CF2, or CF3 by adding spore suspension to make the final concentration of 1 × 104 CFU/g soil. Plants grown in non-infested soil served as controls. There were three replicates per treatment. All the treated pots were placed in a field. After 13 weeks, the same symptoms of dry rot were observed as previously described. No symptoms were observed on the control plants. The trial was repeated. Symptomatic tissues from the inoculated roots were sampled and the pathogen was re-isolated, and identified using PCR. To our knowledge, this is the first report of F. oxysporum and F. solani causing dry rot of carrot in China. References: (1) H. Abe et al. Annual Report of the Society of Plant Protection of North Japan, 48:106-108, 1997. (2) X. Lu. Plant Dis. 97:991, 2013. (3) A. F. Sherf and A. MacNab. Pages 138-139 in: Vegetable Diseases and Their Control. John Wiley & Sons, Inc., 1986.

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First Report of Fusarium oxysporum f. sp. lycopersici Race 3 and F. oxysporum f. sp. radicis-lycopersici in Tomatoes in the Azapa Valley of Chile

Plant Disease ◽

10.1094/pdis-03-14-0303-pdn ◽

2014 ◽

Vol 98 (10) ◽

pp. 1432-1432 ◽

Cited By ~ 4

Author(s):

G. Sepúlveda-Chavera ◽

W. Huanca ◽

R. Salvatierra-Martínez ◽

B. A. Latorre

Keyword(s):

Fusarium Oxysporum ◽

Tap Water ◽

Universal Primers ◽

Conidial Suspension ◽

Laboratory Manual ◽

First Report ◽

Solanum Lycopersicum L ◽

Vascular Discoloration ◽

Seedling Roots ◽

Pcr Assays

Tomato (Solanum lycopersicum L.) is an important crop in the Azapa Valley (18°35′ S, 69°30′ W) in northern Chile, with approximately 600 ha of fresh tomatoes under greenhouses. Cultivars resistant to Fusarium oxysporum f. sp. lycopersici (FOL) races 1 and 2 are mainly used. However, in 2012 and 2013, Fusarium wilt incidence was 2 to 3%. Symptoms appeared unilaterally and consisted of yellowing, leaf wilting of lower leaves, dark brown vascular discoloration, and plant death. The aim of this study was to determine the causal agent of tomato wilt in seven tomato greenhouses in the Azapa Valley. Stem samples (5 × 5 mm) were obtained 10 cm of the stem base from wilted tomatoes ‘Naomi’ (BIOAMERICA S.A., Chile) or from Maxifort tomato rootstock (De Ruiter Seed, USA), both FOL resistant to races 1 and 2. Samples were washed with tap water, surface sterilized with 1% NaClO for 3 min, and incubated on sterile moist paper towels in petri plates for 5 days at 22°C. Mycelial fragments from white colonies, emerging from diseased tissues, were transferred to PDA. Six Fusarium isolates were characterized by the presence of hyaline macroconidia, mostly 3 to 5 septate, slightly curved (19.2 to 32.1 × 2.9 to 4.5 μm) and single-celled, oval to elongated microconidia (3.1 to 8.9 × 2.0 to 4.0 μm). Chlamydospores were single or in pairs. These isolates were identified as F. oxysporum (3). The identity of F. oxysporum was confirmed by PCR assays using genomic DNA of each isolated and the universal primers Uni F and Uni R that generate a 672-bp PCR product. The pathogenic form and races were determined by PCR assays using the specific primers uni, sp13, sp23, and sprl that were able to discriminate all the three FOL races as well as F. oxysporum f. sp. radicis-lycopersici (FORL) isolates (2). The sp13 and sp23 primers amplified DNA bands of 445 and 518 bp, confirming the identity of FOL race 3. However, sprl amplified a fragment of 947 bp corresponding to FORL (2). Pathogenicity tests were conducted on 25-day-old seedlings (10 seedlings per isolate) of tomato ‘Poncho Negro,’ which is susceptible to FOL and FORL. Seedling roots were cut, submerged for 5 min in conidial suspension of 2 × 106 conidia/ml, and transplanted to 250-ml plastic containers with sterile substrate (sand/peat, 1:1). Equally treated non-inoculated seedlings were left as controls. The first symptoms induced by each of the five FOL isolates appeared 8 days after incubation under greenhouse and were characterized by yellowing of older leaves, sometimes affecting one side of the plant, vascular discoloration of the stem, and eventually plant death. In contrast, all seedlings inoculated with a FORL isolate developed a necrotic lesion and vascular discoloration at the base of the stems near the soil line, followed by wilting and plant death. Control plants remained asymptomatic. F. oxysporum was re-isolated only from inoculated plants, completing Koch's postulates. FOL and FORL were reported earlier in other tomato growing areas of Chile (1), located over 1,000 km south of the Azapa Valley. However, this is the first report of FOL race 3 and FORL in the Azapa Valley and FOL race 3 is reported for the first time in Chile. References: (1) S. Acuña. Compendio de Fitopatógenos de Cultivos Agrícolas. Servicio Agrícola y Ganadero. Gobierno de Chile, 2008. (2) Y. Hirano and T. Arie. J. Gen. Plant Pathol. 72:273, 2006. (3) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006.

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First Report of Umbel Browning and Stem Necrosis Caused by Diaporthe angelicae on Carrot in France

Plant Disease ◽

10.1094/pdis-06-13-0673-pdn ◽

2014 ◽

Vol 98 (3) ◽

pp. 421-421 ◽

Cited By ~ 3

Author(s):

L. Ménard ◽

P. E. Brandeis ◽

P. Simoneau ◽

P. Poupard ◽

I. Sérandat ◽

...

Keyword(s):

Seed Production ◽

White Light ◽

Agar Medium ◽

Malt Agar ◽

Universal Primers ◽

Conidial Suspension ◽

First Report ◽

Daucus Carota L ◽

Cropping Seasons ◽

Production Areas

In 2011, carrot (Daucus carota L.) seed production occurred on 2,900 ha, which accounts for approximately 25% of the area devoted to the production of vegetable fine seeds. Since 2007, symptoms of umbel browning have been regularly observed in carrot production areas located in the central region. Initially, triangular necrotic lesions appeared on carrot umbels that later spread to the entire umbels and often progressed to the stems. Diseased umbels became dried prematurely, compromising seed development. The loss in seed production was estimated at approximately 8% of the harvested carrot umbels during the cropping seasons of spring and summer 2007 and 2008 in France. In collaboration with seed companies, diseased carrot stems were collected from seven fields of seed production (eight plants per field) and a fungus was isolated from the tissue. The cultures were grown on malt (2%) agar (1.5%) medium and incubated for 2 weeks at 22°C in darkness. Young fungal colonies were white and a brownish green pigmentation developed when the colonies became older. The same color was observed from the top and on the reverse of the colonies. To induce sporulation, isolates were grown on water agar (1.5%) medium in the presence of carrot stem fragments for 1 week at 22°C in darkness, followed by 1 week at 22°C in white light under a 16-h photoperiod. Pycnidia were produced on stem fragments and contained alpha and beta conidia typical of the genus Diaporthe (2). Alternatively, pycnidia were also obtained on malt agar medium after 2 weeks of culture at 25°C in white light under a 12-h photoperiod. The size of alpha and beta conidia was 6.3 ± 0.5 × 2.3 ± 0.4 μm and 23.3 ± 1.8 × 0.9 ± 0.2 μm, respectively (n = 170). In order to confirm the identification at the genus level and determine the species, DNA was extracted from the mycelium of three representative isolates and the ITS regions of the ribosomal DNA were amplified using universal primers (1). The sequences of the amplified products (GenBank Accession Nos. KF240772 to KF240774) were 100% identical with the ITS sequence of a Diaporthe angelicae isolate deposited in the NCBI database (CBS 111592 isolate, KC343027). To confirm pathogenicity, the three isolates of D. angelicae were inoculated on carrot umbels in the greenhouse. A total of nine plants were inoculated (three plants per isolate). Using a micropipette, 10 μl of a conidial suspension containing alpha and beta conidia (105 conidia mL–1) were deposited at the base of the primary umbel and two secondary umbels, which were wounded before inoculation using a scalpel blade. Seven inoculated plants developed triangular, necrotic lesions that were typical umbel browning. D. angelicae was re-isolated on malt agar medium from the inoculated diseased carrot umbels. To our knowledge, this is the first report of D. angelicae in carrot cultivated for seed production in France. The disease resembles the lesions described in the Netherlands in 1951 on carrot inflorescence caused by Phomopsis dauci (3). In future experiments, it would be crucial to precisely determine if D. angelicae could be transmitted to the seeds. References: (1) M. A. Innis et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990. (2) J. M. Santos and A. J. L. Philips. Fungal Divers. 34:111, 2009. (3) J. A. von Arx. Eur. J. Plant Pathol. 57:44, 1951.

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First Report of Pythium aphanidermatum Causing Stalk Rot on Abelmoschus manihot (L. ) Medic in China

Plant Disease ◽

10.1094/pdis-06-21-1133-pdn ◽

2021 ◽

Author(s):

Qing Qu ◽

Liu Shiwei ◽

Ning Liu ◽

Yunxia Liu ◽

Jia Hui ◽

...

Keyword(s):

Management Practices ◽

Vascular System ◽

Morphological Characteristics ◽

Pythium Aphanidermatum ◽

Negative Control ◽

Perennial Herb ◽

Universal Primers ◽

First Report ◽

Stalk Rot ◽

Aerial Hyphae

Abelmoschus manihot (Linn. ) Medicus (A. manihot) is an annual to perennial herb of the Malvaceae okra, mainly distributed in Guangdong, Guangxi, Fujian, Hunan, Hubei provinces. It can not only be used as an ornamental flower, but also has important economic and medicinal value. Last year, 10% A. manihot in 1,000 acres were observed with stalk rot in the Zhongshang Agricultural Industrial Park, 50 meters east of Provincial Highway 235 in Gaoyang County of Hebei province. Internal discoloration of the stem began brown to black discoloration of the vascular system and became hollow, with the mycelium growing on the surface. Stems from symptomatic plants (approximately 5 mm2) were dissected, washed free of soil, then soaked in 75% ethanol for 16 s to surface-sterilize, and 40 s in HgCl2, then rinsed three times in sterile water. After being dried with blotting paper, five pieces were placed on potato dextrose agar (PDA). After cultured 2 or 3 days, five isolates were purified and re-cultured on PDA in the dark at 25°C. The color of the colony was white. The hyphae were radial in PDA, and the aerial hyphae were flocculent, well-developed with luxuriant branches. The colonies were white and floccus, and the aerial hyphae were well developed, branched and without septum on corn meal agar (CMA). The sporangia were large or petal shaped, composed of irregular hyphae, terminal or intermediate , with the size of (31.6-88.4) μm ×(12.7- 14.6) μm. Vesicles were spherical, terminal or intermediate, ranging from 14.6 to 18.5μm. Oogonia were globose, terminal and smooth which stipe was straight. Antheridia were clavate or baggy and mostly intercalary, sometimes terminal. Oospores were aplerotic, 21.5 to 30.0 μm in diameter, 1.6 to 3.1 μm in wall thickness. The isolates morphological characteristics were consistent with P. aphanidermatum (van der Plaats-Niterink 1981, Wu et al. 2021 ). To identify the isolates, universal primers ITS1/ITS4 (White et al. 1993) were used for polymerase chain reaction–based molecular identification. The amplification region was sequenced by Sangon Biotech (Shanghai, China) and submitted to GenBank (MW819983). BLAST analysis showed that the sequence was 100% identical to Pythium aphanidermatum. Pathogenicity tests were conducted 3 times, with 4 treatments and 2 controls each time. The plants treated were 6 months old. Then the hyphae growing on PDA for 7 days were cut into four pieces. Next, they were inoculated into the soil of the A. manihot. Negative control was inoculated only with PDA for 7 days ( Zhang et al. 2000). The plants were then placed in a greenhouse under 28°C, 90% relative humidity. After inoculated 20 to 30 days, the infected plants showed stalk rot, the same symptoms as observed on the original plants. The control plants didn’t display symptoms. Pythium aphanidermatum was re-isolated from infected stems and showed the same characteristics as described above and was identical in appearance to the isolates used to inoculate the plants. To our knowledge, this is the first report of Pythium aphanidermatum infecting A. manihot stem and causing stalk rot in China. It may become a significant problem for A. manihot. Preliminary management practices are needed for reducing the cost and losses of production.

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First Report of Fusarium incarnatum-equiseti Species Complex Causing Leaf Spot on Rockmelon (Cucumis melo L.) in Malaysia

Plant Disease ◽

10.1094/pdis-06-20-1380-pdn ◽

2020 ◽

Author(s):

Siti Izera Ismail ◽

Nur Ainina Noor Asha ◽

Dzarifah Zulperi

Keyword(s):

Leaf Spot ◽

Cucumis Melo ◽

Species Complex ◽

Fruit Rot ◽

Peat Moss ◽

Distilled Water ◽

Conidial Suspension ◽

Isolation Technique ◽

First Report ◽

Cucumis Melo L

Rockmelon, (Cucumis melo L.) is an economically important crop cultivated in Malaysia. In October 2019, severe leaf spot symptoms with a disease incidence of 40% were observed on the leaves of rockmelon cv. Golden Champion at Faculty of Agriculture, Universiti Putra Malaysia (UPM). Symptoms appeared as brown necrotic spots, 10 to 30 mm in diameter, with spots surrounded by chlorotic halos. Pieces (5 x 5 mm) of diseased tissue were sterilized with 0.5% NaOCl for 1 min, rinsed three times with sterile distilled water, plated onto potato dextrose agar (PDA) and incubated at 25°C for 7 days with a 12-h photoperiod. Nine morphologically similar isolates were obtained by using single spore isolation technique and a representative isolate B was characterized further. Colonies were abundant, whitish aerial mycelium with orange pigmentation. The isolates produced macroconidia with 5 to 6 septa, a tapered with pronounced dorsiventral curvature and measured 25 to 30 μm long x 3 to 5 μm wide. Microconidia produced after 12 days of incubation were single-celled, hyaline, ovoid, nonseptate, and 1.0 to 3.0 × 4.0 to 10.0 µm. Morphological characteristics of the isolates were similar to the taxonomic description of Fusarium equiseti (Leslie and Summerell 2006). Genomic DNA was extracted from fresh mycelium using DNeasy Plant Mini kit (Qiagen, USA). To confirm the identity of the fungus, two sets of primers, ITS4/ITS5 (White et al. 1990) and TEF1-α, EF1-728F/EF1-986R (Carbone and Kohn 1999) were used to amplify complete internal transcribed spacer (ITS) and partial translation elongation factor 1-alpha (TEF1-α) genes, respectively. BLASTn search in the NCBI database using ITS and TEF-1α sequences revealed 99 to 100% similarities with species of both F. incarnatum and F. equiseti. BLAST analysis of these in FUSARIUM-ID database showed 100% and 99% similarity with Fusarium incarnatum-F. equiseti species complex (FIESC) (NRRL34059 [EF-1α] and NRRL43619 [ITS]) respectively (Geiser et al. 2004). The ITS and TEF1-α sequences were deposited in GenBank (MT515832 and MT550682). The isolate was identified as F. equiseti, which belongs to the FIESC based on morphological and molecular characteristics. Pathogenicity was conducted on five healthy leaves of 1-month-old rockmelon cv. Golden Champion grown in 5 plastic pots filled with sterile peat moss. The leaves were surface-sterilized with 70% ethanol and rinsed twice with sterile-distilled water. Then, the leaves were wounded using 34-mm-diameter florist pin frog and inoculated by pipetting 20-μl conidial suspension (1 × 106 conidia/ml) of 7-day-old culture of isolate B onto the wound sites. Control leaves were inoculated with sterile-distilled water only. The inoculated plants were covered with plastic bags for 5 days and maintained in a greenhouse at 25 °C, 90% relative humidity with a photoperiod of 12-h. After 7 days, inoculated leaves developed necrotic lesions similar to the symptoms observed in the field while the control treatment remained asymptomatic. The fungus was reisolated from the infected leaves and was morphologically identical to the original isolate. F. equiseti was previously reported causing fruit rot of watermelon in Georgia (Li and Ji 2015) and China (Li et al. 2018). This pathogen could cause serious damage to established rockmelon as it can spread rapidly in the field. To our knowledge, this is the first report of a member of the Fusarium incarnatum-F.equiseti species complex causing leaf spot on Cucumis melo in Malaysia.

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First report of charcoal rot caused by Macrophomina phaseolina on potato tubers in Mauritius

Plant Disease ◽

10.1094/pdis-02-21-0258-pdn ◽

2021 ◽

Author(s):

Sandhya Devi Takooree ◽

Hudaa Neetoo ◽

Mala Ranghoo-Sanmukhiya ◽

Jacquie vander Waals ◽

Mira Vojvodić ◽

...

Keyword(s):

Sodium Hypochlorite ◽

Tap Water ◽

Solanum Tuberosum L ◽

Pcr Amplification ◽

Its Region ◽

Macrophomina Phaseolina ◽

Negative Control ◽

Potato Tubers ◽

Charcoal Rot ◽

First Report

Charcoal rot, caused by Macrophomina phaseolina, is an important disease in tropical and subtropical regions which affects a broad range of host plants, including potato (Solanum tuberosum L.). In this crop, charcoal rot can reduce the marketable quality of tubers (Arora 2012) and cause yield losses up to 88% (Somani 2007). During a survey of a potato field of ‘Spunta’ cultivar in Goodlands, Mauritius (20°02'28.2"S 57°39'30.4"E) approximately 10% of tubers with grey pigmentation around the lenticels and small water-soaked spots with white dots were observed. These symptoms later advanced to dark brown to black patches on the skin surface, all conforming to typical symptoms of charcoal rot (Arora and Khurana 2004). Fragments of infected and adjacent healthy tissue were cut, thoroughly washed with tap water, surface sterilized for 30 s with 1% sodium hypochlorite (25% bleach), placed on chloramphenicol-amended Potato Dextrose Agar (PDA), and incubated for 5 days in the dark at 25±2oC. From all the inoculated plates, only fast-growing, dark brown, grey to black Macrophomina-like colonies grew and several mono-sclerotial isolates were obtained with uniform morphological features. Following staining with cotton lactophenol dye using the clean slide technique, the isolate 449G-19/M exhibiting typical characteristics of M. phaseolina (Arora and Dhurwe 2019) and forming flattened, globose, black sclerotia with an average diameter of 180 µm (n= 50), was selected and used for further characterization. Identification was confirmed by sequencing of the ITS region of rDNA. Total DNA was extracted directly from the mycelium using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions, while PCR amplification and sequencing were performed with primers ITS1-F (Gardes and Bruns 1993) and ITS-4 (White et al. 1990). The nucleotide sequence of the isolate 449G-19/M (Accession No. MW301138) shared 98.28 to 99.80% similarity with over 70 M. phaseolina isolates in GenBank (99.18% with isolate from Zea mays, Accession No. KF531825 (Phillips et al. 2013)). Pathogenicity was tested on 20 healthy tubers which were initially disinfected with 2% sodium hypochlorite for 1 min and individually placed in pots (20 cm ø) containing sterile substrate. Ten tubers were inoculated by placing colony fragments of 7-day-old cultures of the isolate 449G-19/M near each tuber. Similarly, 10 tubers inoculated with sterile PDA served as a negative control. The plants were maintained in greenhouse conditions, watered daily, and assessed for the presence of symptoms 8 weeks post emergence. All inoculated tubers exhibited charcoal rot on progeny tubers while control plants remained symptomless. Koch’s postulates were fulfilled successfully and the fungus recovered from the inoculated plants. Although M. phaseolina was previously observed in Mauritius on groundnut resulting in pre-emergence rot and collar rot (Anonymous 1962), to our knowledge, this is the first report demonstrating charcoal rot on potato tubers caused by M. phaseolina in Mauritius. As the sclerotia can remain in the soil for long periods of time (Arora and Khurana 2004) and with prevailing conditions of global warming, charcoal rot may be a threat for potatoes and other local crops (Somani et al. 2013). This study will sensitize agricultural extension officers on this new disease and calls for routine surveillance to safeguard this crop.

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First Report of Campylocarpon fasciculare Causing Black Foot Disease of Grapevine in Spain

Plant Disease ◽

10.1094/pdis-02-11-0105 ◽

2011 ◽

Vol 95 (8) ◽

pp. 1028-1028

Author(s):

S. Alaniz ◽

C. Agustí-Brisach ◽

D. Gramaje ◽

M. I. Aguilar ◽

A. Pérez-Sierra ◽

...

Keyword(s):

Tap Water ◽

Peat Moss ◽

Vitis Vinifera L ◽

Sodium Hypochlorite Solution ◽

Distilled Water ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Foot Disease ◽

Black Foot ◽

Black Foot Disease

In May 2008, symptoms of black foot disease were observed on 8-year-old grapevines (Vitis vinifera L.) cv. Garnacha in Albuñol (Granada Province, southern Spain). Affected plants showed delayed budding with low vigor. Roots showed black discoloration and necrosis of wood tissues. Root fragments were cut, washed under running tap water, surface sterilized for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. Small pieces of discolored or necrotic tissues were plated onto potato dextrose agar (PDA) supplemented with 0.5 g liter–1 of streptomycin sulfate. Plates were incubated at 25°C in the dark for 10 days and all colonies were transferred to PDA. A Cylindrocarpon-like fungus was consistently isolated from necrotic root tissues. Single conidial isolates were obtained and grown on PDA and Spezieller Nährstoffarmer Agar (SNA) and incubated at 25°C for 10 days in darkness. On PDA, the isolates developed white, thick, and cottony to felty abundant mycelium. On SNA, all isolates produced slightly to moderately curved one-septate (22.5-) 25.6 (-27.5) × (5-) 5.63 (-6.25) μm, two-septate (30-) 36.1 (-45) × (6.25-) 7.08 (-7.5) μm, three-septate (37.5-) 47.9 (-52.5) × (6.25-) 7.5 (-8.75) μm, four-septate (47.5-) 53.3 (-62.5) × (7.5-) 7.89 (-8.75) μm, and five-septate (52.5-) 61.8 (-67.5) × (7.5-) 8 (-8.75) μm macroconidia. Microconidia were not observed. DNA sequence of the rDNA internal transcribed spacer region (ITS) was obtained for isolate Cf-270 and deposited in GenBank (Accession No. HQ441249). This sequence showed high similarity (99%) to the sequence of Campylocarpon fasciculare Schroers, Halleen & Crous (GenBank Accession No. AY677303), in agreement with morphological features (1). Pathogenicity tests were conducted with inoculum produced on wheat (Triticum aestivum L.) seeds that were soaked for 12 h in flasks filled with distilled water. Each flask contained 300 ml of seeds that were subsequently autoclaved three times after excess water was drained. Two fungal disks of a 2-week-old culture of C. fasciculare (isolate Cf-270) grown on PDA were placed aseptically in each flask. The flasks were incubated at 25°C for 4 weeks and shaken once a week to avoid clustering of inoculum. Plastic pots (220 cm3) were filled with a mixture of sterilized peat moss and 10 g of inoculum per pot. One-month-old grapevine seedlings were planted individually in each pot and placed in a greenhouse at 25 to 30°C in a completely randomized design. Control plants were inoculated with sterile uninoculated seeds. Six replicates (each one in individual pots) were used, with an equal number of control plants. The experiment was repeated. Symptoms developed on all plants 20 days after inoculation and consisted in reduced vigor, interveinal chlorosis and necrosis of the leaves, necrotic root lesions with a reduction in root biomass, and plant death. The fungus was reisolated from the roots of affected seedlings and identified as C. fasciculare, completing Koch's postulates. No symptoms were observed on the control plants. Black foot disease of grapevines can be caused by different species of Cylindrocarpon and Campylocarpon. C. fasciculare was first reported in South Africa in 2004 (1). To our knowledge, this is the first report of C. fasciculare causing black foot disease of grapevine in Spain as well as other countries in Europe. Reference: (1) F. Halleen et al. Stud. Mycol. 50:431, 2004.

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First Report of Damping-Off Caused by Cylindrocarpon pauciseptatum on Pinus radiata in Spain

Plant Disease ◽

10.1094/pdis-02-11-0125 ◽

2011 ◽

Vol 95 (7) ◽

pp. 874-874 ◽

Cited By ~ 6

Author(s):

C. Agustí-Brisach ◽

A. Pérez-Sierra ◽

F. García-Figueres ◽

C. Montón ◽

J. Armengol

Keyword(s):

Pinus Radiata ◽

Tap Water ◽

Morphological Characteristics ◽

Peat Moss ◽

Sodium Hypochlorite Solution ◽

Distilled Water ◽

Damping Off ◽

First Report ◽

Wheat Kernels ◽

Needle Blight

In the fall of 2009, damping-off of Pinus radiata seedlings was observed in a pine nursery in Sant Feliu de Buixalleu, Girona Province, northeastern Spain. Plants exhibited needle blight, extensive root necrosis, and root death. Root sections of symptomatic plants were cut, washed under running tap water, surface disinfected for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. Small fragments of discolored tissues were plated onto potato dextrose agar (PDA) supplemented with 0.5 g liter–1 of streptomycin sulfate. Plates were placed at 25°C in the dark for 10 to 14 days, and all fungal colonies were transferred to PDA. A Cylindrocarpon sp. was consistently isolated from necrotic root tissues. Single-conidial isolates were obtained and grown on PDA and Spezieller Nährstoffarmer agar (SNA) (2) at 25°C for 10 days with a 12-h photoperiod. On PDA, the isolates developed abundant mycelium, which varied from white-to-grayish brown or golden brown. On SNA, all isolates produced two-septate, (35-) 39.4 (-40) × (7.5-) 7.7 (-8.75) μm, and three-septate, (32.5-) 40.9 (-52.5) × (7.5-) 7.7 (-8.75) μm, macroconidia. Microconidia, one-septate macroconidia, and chlamydospores were not observed. Identity of these isolates was determined by a multiplex PCR system using a set of three pair of specific primers (Mac1/MaPa2, Lir1/Lir2, and Pau1/MaPa2) (1), which generated a 117-bp product that was characteristic of Cylindrocarpon pauciseptatum Schroers & Crous. Morphological characteristics also supported this identification (4). Internal transcribed spacers regions (ITS1 and ITS4) of rDNA were obtained for isolate 1052 and deposited in GenBank (Accession No. HQ441248). This sequence was identical (100%) with the sequence of C. pauciseptatum (GenBank Accession No. HM036590). Pathogenicity tests were conducted with inoculum produced on wheat kernels that were soaked in distilled water in flasks for 12 h. Each flask contained 200 ml of kernels that were subsequently autoclaved three times after excess water was drained. Two fungal disks from a 2-week-old culture of C. pauciseptatum (isolate 1052) grown on PDA were placed aseptically in each flask. Cultures in flasks were incubated at 25°C for 4 weeks and shaken once a week. A plastic pot (220 cm3) was filled with a mixture of sterilized peat moss and 10 g of inoculum. A 1-month-old seedling of P. radiata was planted in plastic pots and placed in a greenhouse at 25 to 30°C in a completely randomized design with six replications. Controls contained sterile wheat kernels. The experiment was repeated. Symptoms developed 20 days after inoculation and consisted of root lesions, a reduction in root biomass, needle blight, and the death of all seedlings. The fungus was reisolated from affected seedlings. Damping-off was not observed on the control plants. C. pauciseptatum causing black foot disease of grapevine (3) was first found in Spain in 2008, but to our knowledge, this is the first report of C. pauciseptatum causing damping-off of P. radiata in Spain. References: (1) S. Alaniz et al. Plant Dis. 93:821, 2009. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, 2006. (3) M. T. Martin et al. Plant Dis. 95:361, 2011. (4) H. J. Schroers et al. Mycol. Res. 112:82, 2008.

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Fungicide Sensitivity of Pythium spp. Associated with Cavity Spot of Carrot in California and Michigan

Plant Disease ◽

10.1094/pdis-07-11-0562 ◽

2012 ◽

Vol 96 (3) ◽

pp. 384-388 ◽

Cited By ~ 7

Author(s):

Xiao Hong Lu ◽

R. Michael Davis ◽

S. Livingston ◽

J. Nunez ◽

Jianjun J. Hao

Keyword(s):

United States ◽

Growth Inhibition ◽

Active Ingredient ◽

Mycelial Growth ◽

The United States ◽

Fungicide Sensitivity ◽

First Report ◽

Cavity Spot ◽

Pythium Violae

The identity of 172 isolates of Pythium spp. from cavity spot lesions on carrot produced in California and Michigan was determined, and their sensitivity to three fungicides was examined. Pythium violae accounted for 85% of California isolates, with P. irregulare, P. dissotocum (the first report as a carrot pathogen in the United States), P. ultimum, and P. sulcatum making the balance. P. sulcatum, P. sylvaticum, and P. intermedium were the most commonly recovered (85%) species in Michigan; others from Michigan included P. intermedium, P. irregulare, and an unclassified strain, M2-05. On fungicide-amended media, 93% of isolates were sensitive to mefenoxam (inhibition of mycelial growth was >60% at 10 μg active ingredient [a.i.]/ml); however, two of five isolates of P. irregulare from California were highly resistant (≤60% inhibition at 100 μg a.i./ml); about half of the isolates of P. intermedium and P. sylvaticum and a single isolate of P. violae were highly or intermediately resistant to mefenoxam (>60% inhibition at 100 μg a.i./ml, or ≤60% inhibition at 10 μg a.i./ml). P. dissotocum, P. irregulare, P. sulcatum, M2-05, and three of seven isolates of P. intermedium were insensitive to fluopicolide (effective concentrations for 50% growth inhibition [EC50] were >50 μg a.i./ml), while P. sylvaticum, P. ultimum, P. violae, and some isolates in P. intermedium were sensitive (EC50 < 1 μg a.i./ml). All isolates were sensitive to zoxamide (EC50 < 1 μg a.i./ml). Sensitivity baselines of P. violae to zoxamide and fluopicolide were established.

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Evaluation of coconut water neutralized by different agents on the viability of human fibroblasts: an in vitro study

Revista de Odontologia da UNESP ◽

10.1590/1807-2577.09216 ◽

2016 ◽

Vol 45 (4) ◽

pp. 234-239 ◽

Cited By ~ 1

Author(s):

Priscilla Barbosa Ferreira SOARES ◽

Aletheia Moraes ROCHA ◽

Manuella Verdinelli de Paula REIS ◽

Camilla Christian Gomes MOURA ◽

Carlos José SOARES

Keyword(s):

Cell Viability ◽

Tap Water ◽

Human Fibroblasts ◽

In Vitro Study ◽

Positive Control ◽

Negative Control ◽

Coconut Water ◽

Ph Adjustment ◽

Adjustment Method

Abstract Objective This study evaluated four types of pH adjustment of the coconut water (CW) on viability of human fibroblasts (HFF). Material and method Natural and industrialized CW were adjusted to pH 7.0 using: (1) Sodium Hidroxide (NaOH), (2) Sodium bicarbonate (NaHCO3), (3) Triethanolamine (C6H15NO3), (4) 2-Amino-2-Methil-1-Propanol (C4H11NO). Fibroblasts were plated at 2×104/ well in 96 well plates and maintained in the CW solutions for 2 h and 4 h. Positive control was represented by HFF maintained in DMEM and the negative control by tap water. Cell viability was analyzed by MTT formazan method. Data were analyzed by 3-way ANOVA followed by Tukey’s and Dunnet’s test. Result There are no significant effect on the cell viability regarding type of CW, period of evaluation, and the interactions between CW and period of evaluation, CW and pH adjustment method, pH adjustment method and period of evaluation (p>0.05). Conclusion The product used for CW pH adjustment did not influenced HFF viability, thought there are a tendency of better performance in natural CW.

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