Sterile Toothpicks Research Articles

Rhizopus arrhizus (syn. R. oryzae) Causing Sunflower Head Rot in Hebei Province, China

<i>Rhizopus arrhizus</i> (syn. <i>R. oryzae</i>) Causing Sunflower Head Rot in Hebei Province, China

First Report of Colletotrichum siamense and C. gloeosporioides Causing Anthracnose of Citrus spp. in Mexico.

Citrus anthracnose, caused by Colletotrichum spp., is a major disease in many citrus-growing regions of the world. During the spring of 2019, symptoms of petal necrosis and necrotic lesions on fruits were detected on Mexican lime (Citrus aurantifolia), sweet orange (Citrus sinensis), and grapefruit (Citrus paradisi) trees in three commercial orchards distributed in northern Sinaloa (El Fuerte and Ahome municipalities), Mexico. Colletotrichum-like colonies were consistently isolated on potato dextrose agar (PDA) medium from symptomatic petals and fruits, and 30 monoconidial isolates (10 per orchard) were obtained. Five isolates were selected as representative for morphological characterization, multilocus phylogenetic analysis, and pathogenicity tests. The isolates were designated as FAVF355-FAVF359 and were deposited in the Culture Collection of Phytopathogenic Fungi of the Faculty of Agronomy of El Fuerte Valley at the Autonomous University of Sinaloa (Mexico). Colonies grown on PDA at 25ºC were cottony, dense, with grayish white aerial mycelium and with pink conidial masses. Conidia (n= 100) were cylindrical, hyaline, aseptate, 13.7 to 18.8 × 4.3 to 5.8 μm, with both ends rounded. Based on morphological features, the five isolates were tentatively identified in the Colletotrichum gloeosporioides species complex (Weir et al. 2012). For molecular identification, total DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), and partial sequences of actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin (TUB2) genes were amplified by PCR (Weir et al. 2012), and sequenced. A phylogenetic tree based on Bayesian inference for species belonging to the C. gloeosporioides species complex was constructed. The multilocus phylogenetic analysis distinguished the isolates FAVF355-FAVF357 as C. gloeosporioides sensu stricto and the isolates FAVF358-FAVF359 as C. siamense. The sequences were deposited in GenBank (accession numbers ITS: MT850050-MT850054; ACT: MT834528-MT834532; GAPDH: MT855979-MT855982; TUB2: MT834533-MT834536). Pathogenicity of the five isolates was verified on healthy fruits of their original host species. Five fruits per isolate were inoculated using the colonized agar plug method. Fruits were wounded with a sterile toothpick and mycelial plugs (5 mm in diameter) removed from the margin of a 6-days-old culture were placed onto three wound sites in each fruit. Non-colonized agar plugs were placed on the wounds of 10 fruits used as the control. The fruits were kept in a moist chamber at 25°C for 8 days. The experiment was repeated twice. All inoculated fruits developed circular and necrotic lesions 6 days after inoculation, whereas the control fruits remained symptomless. The fungi were consistently re-isolated from the diseased fruits and were morphologically identical to that originally inoculated, fulfilling Koch´s postulates. To date, only C. gloeosporioides sensu lato and C. acutatum sensu lato has been associated with sweet orange and Mexican lime in Mexico (Farr and Rossman 2020), whereas C. gloeosporioides sensu stricto has been recently recorded in a different area (Iguala, Guerrero) of Mexico (Cruz-Lagunas et al. 2020). To our knowledge, this is the first report of C. gloeosporioides sensu stricto causing anthracnose on sweet orange, and of C. siamense on Mexican lime in Mexico, as well as C. gloeosporioides s. s. causing disease on grapefruit in Sinaloa, Mexico.

First Report of Colletotrichum jiangxiense Causing Avocado Anthracnose in Mexico.

Mexico is the largest avocado (Persea americana) producer and exporter in the world. In January of 2019, typical symptoms of fruit anthracnose were observed on approximately 90% of avocado trees in backyards localized in Leonardo Bravo municipality in Guerrero, Mexico. Lesions on avocado fruits were circular, necrotic, and sunken, whereas the mesocarp showed a soft rot with dark brown discoloration. To perform fungal isolation, small pieces from adjacent tissue to lesions of five symptomatic fruits were surface disinfested by immersion in a 1% sodium hypochlorite solution for 2 min, rinsed in sterile distilled water, and placed in Petri dish containing potato dextrose agar (PDA). Plates were incubated at 25 ºC for 5 days in darkness. Colletotrichum-like colonies were consistently isolated and seven monoconidial isolates were obtained. An isolate was selected as a representative for morphological characterization, molecular analysis, and pathogenicity tests. The isolate was deposited in the Culture Collection of Phytopathogenic Fungi at the Colegio Superior Agropecuario del Estado de Guerrero (Accession No. CSAEG-CJ19). After 8 days on PDA, the colonies were gray on the upper surface, and with orange conidial masses. Conidia (n= 100) were cylindrical, hyaline, aseptate, with rounded ends, 14.4 to 18.5 × 4.5 to 6.2 μm. Based on morphological features, the isolate was tentatively identified in the C. gloeosporioides species complex (Weir et al. 2012). For molecular identification, genomic DNA was extracted and the internal transcribed spacer (ITS) region of rDNA, and partial sequences of actin (ACT), β-tubulin (TUB2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified by PCR, and sequenced with primers ITS5/ITS4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn 1999), Bt2A/Bt2B (Glass and Donaldson 1995), and GDF/GDR (Templeton et al. 1992), respectively. BLAST analysis of the obtained sequences of the ITS, ACT, TUB2, and GAPDH genes revealed 100%, 99.63%, 99.77% and 100% identity with those of isolate LF687 of C. jiangxiense in GenBank (Accession numbers KJ955201, KJ954471, KJ955348, and KJ954902). A phylogenetic tree based on Bayesian inference and including published ITS, ACT, TUB2, and GAPDH data for Colletotrichum species was constructed. The multilocus phylogenetic analysis clearly distinguished the isolate CSAEG-CJ19 as C. jiangxiense separating it from all other species within the C. gloeosporioides species complex. The sequences were deposited in GenBank (accession numbers ITS:MT011397; ACT:MN968784, TUB2:MN968786, and GAPDH:MN968785). To conduct Koch's postulates, 20 healthy avocado fruits (cv. Hass) were wounded with a sterile toothpick (2 mm in depth) and a drop of 15 µl of conidial suspension (1 × 105 spores/mL) was placed on each wound. Ten control fruit were wounded and treated with sterilized water. All the fruits were kept in a moist plastic chamber at 25°C for 8 days. All inoculated fruits developed circular and necrotic lesions (12 to 18 mm in diameter), 5 days after inoculation, whereas control fruits remained healthy. The fungus was consistently re-isolated from the inoculated fruits. Previously, C. jiangxiense has been reported as a pathogen on Camellia sinensis and Citrus sinensis in China (Farr and Rossman 2020). To our knowledge, this is the first report of C. jiangxiense causing anthracnose on avocado worldwide. This study shown another species in the C. gloeosporioides complex associated with avocado diseases in Mexico. Therefore, it is necessary to explore the diversity of Colletotrichum species in detail through subsequent phylogenetic studies as well as to monitor the distribution of this pathogen into other Mexican regions.

First Report of Anthracnose Fruit Rot Caused by Colletotrichum fioriniae on Red Raspberry (Rubus idaeus) in the Mid-Atlantic Region of the United States.

Colletotrichum fioriniae Marcelino & Gouli ex R.G. Shivas & Y.P. Tan causes anthracnose and fruit rot in various hosts worldwide (Shivas and Tan 2009). In June 2019, 40% of red raspberry fruit (cv. Prelude) in a plot at the Wye Research and Education Center of the University of Maryland in Queenstown, MD, showed light to dark brown, rotten, and slightly sunken drupelets. Symptomatic berries (n = 17) were collected and incubated in plastic boxes at room temperature (22°C) to obtain 41 single-spore isolates. Colonies on potato dextrose agar (PDA) were gray to pale olivaceous, cottony, with aerial mycelium in compact tufts, and had a white rim and a pinkish-red to pale orange or yellow reverse and orange acervuli. Conidia were one-celled, hyaline, smooth-walled, with either both acute ends or one round and one pointed end, without setae. Conidial dimensions were recorded for two single-spore isolates and were in accordance with previous measurements (Damm et al. 2012): mean length ± SD × width ± SD = 12.9 ± 1.5 × 5.4 ± 0.9 μm on PDA (n = 60). DNA was extracted from mycelium of 11 single-spore isolates (Chi et al. 2009). Damm et al. (2012) showed that all subclades of the Colletotrichum acutatum complex can be identified by amplifying a 290-bp section of the glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene (primers: Templeton et al. [1992]) and a 1,440-bp section of the β-tubulin-2 gene (tub2) (primers: T1 from O’Donnell and Cigelnik [1997], TubR1 from Hu et al. [2015]). Polymerase chain reaction products were sequenced from two directions. Three consensus sequences for each gene were deposited into GenBank: RB19-14.4S (MN520488, MN532086), RB19-13.3S (MN520489, MN532087), and RB19-26P (MN520490, MN532088). BLASTn search confirmed 100% identity with the Colletotrichum fioriniae sequences JQ949974 (491 bp, tub2) and JQ948653 (254 bp, gapdh) for RB19-14.4S and in total nine of the 11 isolates. For isolate RB19-26P the gapdh sequence (MN532088) was 100% identical with JQ948624 instead. Tub2 (MN520489) and gapdh (MN532087) sequences for isolate RB19-13.3S were 100 and 99.6% identical with C. fioriniae sequences JQ949944 and JQ948623, respectively. To confirm pathogenicity, healthy ripe red raspberries (cv. Jaclyn) were inoculated with a mixture of four single-spore isolates. Berries were surface sterilized by washing in 0.1% Tween 20 for 2 min, 70% ethanol for 30 s, 1% NaOCl for 1 min, and sterile water for 2 min. After air drying for 2 h, berries were either wounded with a sterile toothpick or not wounded and were inoculated with a 15-μl drop of spore suspension (5 × 10⁵ conidia/ml) or sterile water. Each treatment was replicated four times with eight berries per replicate. The experiment was conducted twice. After incubation for 3 days, both wounded and unwounded inoculated berries showed symptoms identical to those described above, and some lesions had orange acervuli. C. fioriniae was reisolated from inoculated berries but never from water-inoculated berries. Identity of the recovered isolates was confirmed morphologically and molecularly. Although C. fioriniae is an endophyte in various plant species worldwide (Freeman et al. 2001) and was previously isolated as an endophyte or from leaves of Rubus idaeus in New York (U.S.A.), Norway, and Mississippi (U.S.A.) (Marcelino et al. 2009; Sundelin et al. 2015; Zhang et al. 2016), it has not been described as a raspberry fruit pathogen causing anthracnose disease so far. To our knowledge this is the first report of C. fioriniae found on red raspberries in the Mid-Atlantic region of the United States.

First Report of Dickeya fangzhongdai Causing Soft Rot of Onion in New York State

In early July 2014, an outbreak of soft rot disease of onion (Allium cepa L.) occurred in Orange County, NY. Symptoms developed following rainy, hot, humid weather and included chlorosis, maceration of leaves, browning, and plant collapse within a few days. Bulbs from diseased plants appeared normal, but inner tissues were macerated. Disease was first apparent in a field of red onions (cv. Redwing); later yellow onions (cultivar unknown) in an adjacent field were symptomatic. Standard techniques were used to isolate bacteria from diseased red onion bulbs (Bonasera et al. 2017). Briefly, 14 onion bulbs were cut open with a disinfected knife, and diseased tissues were probed with sterile wooden applicators and streaked on onion extract medium agar (Zaid et al. 2012). Plates were incubated for 2 days at 28°C, and individual colonies were streaked to purity on Luria–Bertani (LB) agar. Small white bacterial colonies with irregular margins were obtained as the predominant type from three samples. A representative colony was identified as Dickeya sp. based on the sequence of a portion of its 16S rRNA gene amplified by fD1/rP2 (Weisburg et al. 1991). Several more colonies were assessed using ADE1 and ADE2 PCR primers specific to Dickeya genus (Nassar et al. 1996), which also indicated that these isolates belonged to the genus Dickeya. Genomic DNA of one of these colonies, AP6, was extracted and sequenced using an Illumina NextSeq500 by the Institute of Biotechnology at Cornell University. Raw reads were processed (Ma et al. 2019) and used for de novo assembly with SPAdes 1.12. The draft genome assembly resulted in 260 contigs, with an N50 size of 50,007 bp, totaling 5.11 Mbp, and it was deposited in GenBank as accession number VS. We conducted average nucleotide identity and in silico DNA-DNA hybridization tests to determine the species of Dickeya strain AP6 using Python package pyANI and GGDC (https://ggdc.dsmz.de), respectively. These analyses indicated that AP6 belongs to species Dickeya fangzhongdai. Koch’s postulates were completed with D. fangzhongdai AP6. Two leaves each of three 80-day-old, healthy-appearing, controlled environment-grown onion plants (cv. Fortress for the first replicate, cv. Redwing for the second and third replicates) were inoculated with D. fangzhongdai AP6. The leaves were first punctured with sterile toothpicks, and then the wound was smeared with bacteria grown overnight on LB agar. One plant was punched with sterile toothpicks as a negative control. Elongated lesions with water-soaking at the margins were observed around the inoculation sites after incubating for 24 h at 30°C; no symptoms were observed on control plants. Disease symptoms progressed very rapidly; after 72 h, all AP6-inoculated plants had collapsed completely, and foliage was quite water-soaked. The experiment was conducted three times. We isolated three isolates from water-soaked lesions 24 h after inoculation and used dnaX sequencing on one of the reisolated colonies to confirm that bacteria in the diseased tissue were identical to the D. fangzhongdai inoculum. D. fangzhongdai was first identified in China as causing bleeding canker necrosis on pear (Tian et al. 2016). D. fangzhongdai was reported to cause soft rot in Welsh onion in Taiwan (Tsai et al. 2019), diseased Phalaenopsis orchids in Slovenia and Aglaonema in St. Lucia, and was found in water samples from Scotland and Malaysia (Alic et al. 2018; Pritchard et al. 2013). Soft rot disease of onion was reported in the United States (Ceponis et al. 1986), but its causal organism had not been investigated thoroughly. Here we report that D. fangzhongdai caused an outbreak of soft rot of onions in Orange County, NY.

First Report of Necrotic Disease Caused by Pantoea agglomerans on Plum (Prunus salicina) in China

Prunus salicina L., commonly called Chinese plum or Japanese plum, is a small deciduous tree native to China and widely distributed in the world. Since spring of 2018, an unknown disease has affected commercial plums grown in an orchard located in Dazhou, Sichuan Province, China (31°40′41″N, 107°43′57″E). Disease symptoms in plum trees include water-soaked lesions on immature fruits, brown necrotic spots, and holes with a yellow halo in leaves, as well as dark brown oily cankers on twigs. To isolate the causal agent, surface-disinfested tissues from three separate symptomatic fruits, leaves, and twigs were placed onto nutrient agar (NA) plates and were incubated at 28°C. Pale yellow colonies with smooth margins and mucoid texture appeared after 12 h of incubation. Individual colonies were transferred two times to NA plates using the conventional streak plate techniques to obtain pure cultures. Purified bacteria cells were rod shaped, 1.5 to 3.0 μm long, and 0.5 to 1.0 μm wide. Three isolates (named CdjP1, CdjP2, and CdjP3) were used for further characterization. Biochemical tests using Gram stain, MacConkey agar, and Kovacs’s indole kit (Solarbio, Beijing, China) indicated that the three isolates were gram negative, lactose positive, and indole negative. The 16s rDNA fragment was amplified for the three strains using universal primers 27F and 1492R (Lane 1991) and sequenced (GenBank accession nos. MN542182, MN542183, and MN542184). BLASTn analysis revealed the nearly full-length (about 1,400 bp) 16S rDNA sequences of CdjP1, CdjP2, and CdjP3 shared over 99% identity with Pantoea agglomerans (MH101508) (identities 1,400/1,406, 1,407/1,412, 1,405/1,413), P. vagans (CP038853) (identities 1,397/1,406, 1,403/1,412, 1,402/1,414), and P. ananatis (KC178592) (identities 1,399/1,406, 1,405/1,412, 1,404/1,414), respectively. Multilocus sequence analysis has been shown to be a powerful molecular method for delineation of Pantoea species (Deletoile et al. 2009; Tambong et al. 2014). To identify the species of CdjP1, CdjP2, and CdjP3, six housekeeping genes (fusA, gyrB, leuS, pyrG, rlpB, and rpoB) of each strain were amplified and sequenced (GenBank accession nos. MN549363 to MN549380). Then a phylogenetic tree was constructed using Bayes inference methods based on fusA-gyrB-leuS-pyrG-rlpB-rpoB concatenated fragments (2,997 bp), which showed that CdjP1, CdjP2, and CdjP3 are most closely related to P. agglomerans. To fulfill Koch’s postulates, pathogenicity tests were conducted on 3-year-old plum trees (cv. Qinba) grown in Sichuan Province, China (31°40′53″N, 107°44′2″E). Immature plum fruits, leaves, and twigs were wounded with a sterile toothpick and were inoculated with 20 µl of bacterial suspension (10⁸ CFU/ml) of each isolate. Plants inoculated with sterile water were used as a control. All treatments and the control were repeated three times. The trees were then grown under natural conditions (monthly mean maximum temperature 32°C, monthly mean minimum temperature 23°C). After 2 weeks, all test-isolate-inoculated plum fruits, leaves, and twigs developed symptoms similar to those of the natural infections observed. No lesions were observed on the controls. Bacteria reisolated from the infected plum fruits, leaves, and twigs were similar in colony appearance to the original isolates, and the 16s rDNA fragments were identical to their original isolate sequences, thereby fulfilling Koch’s postulates. Based on colony morphology, biochemical tests, 16S rRNA gene sequencing data, and phylogenetic analysis, the pathogen was identified as P. agglomerans. In China, P. agglomerans has been reported to infect cotton, walnut, Vigna angularis, and Ziziphus jujuba (Lu et al. 2015; Ren et al. 2008; She et al. 2019; Yang et al. 2011). To our knowledge, this is the first report of P. agglomerans causing bacterial necrosis of plum in China or worldwide. This report expands the host range of P. agglomerans and will help to develop effective disease control strategies, as the etiology is known.

Fusarium Wilt of Imperial Chrysanthemum (Chrysanthemum morifolium) Caused by Fusarium oxysporum in China

Imperial chrysanthemum (Chrysanthemum morifolium) is cultivated in China as medicinal plant. In October 2018 at Yunfu in Guangdong province, wilted stems, necrotic leaves, and root rot were observed on many imperial chrysanthemum plants. The incidence of disease was up to 50%. Infected plants were collected for further examination and fungal isolation. Small pieces of symptomatic tissues were surface sterilized with 75% alcohol for 10 s followed by 2.5% NaClO for 15 s, rinsed in sterile water three times, air dried, and transferred onto potato dextrose agar medium at 25°C for 7 days. Pure cultures were obtained by isolating single hyphae. Colonies were initially white, but after about 17 days, the center of the colony began to turn purple. The colonies produced were hyaline. Microconidia were single-celled, ellipsoid to cylindrical, and 3.1 to 5.4 × 1.3 to 2.7 μm. Fusiform macroconidia were produced on carnation leaf agar, which were mostly three-septate, 12.5 to 24.0 × 2.0 to 3.8 μm, with a prominent foot-shaped basal cell. The observed traits were identical to those reported for other Fusarium species (Leslie and Summerell 2006). Genomic DNA was extracted from isolated colonies and amplified with three pairs of primers: universal primers ITS1/ITS4 (White et al. 1990), EF1/EF2 (Geiser et al. 2004), and FO1/FO2 (Fernandez-Ortuno et al. 2010). Isolates were identified to species by comparing the DNA sequences generated using BLAST against sequences in GenBank’s nucleotide database, Fusarium-ID, and Fusarium MLST. The 558-bp ITS sequence (GenBank MK385594) showed 100% identity to Fusarium oxysporum (GenBank MH911375 and SH1610159.08 FU JQ039192). The 712-bp TEF1α (GenBank MK404060) showed 99.85% identity to F. oxysporum (FD_00576), 100% identity to F. oxysporum (GenBank MG670365, JF430176, and SH1610159.08 FU JQ039192). PCR primers FO1/FO2 amplified a 699-bp cytochrome P450 (GenBank MK404061) that showed 100% identity to F. oxysporum (GenBank QDZ38461) by BLASTx in the NCBI. Based on the morphological characteristics and nucleotide homology, the isolate was identified as F. oxysporum. To satisfy Koch’s postulates, a pathogenicity experiment was conducted by using a spore suspension. Healthy plants’ roots were wounded with a sterile toothpick and soaked in spore suspension (∼1 × 10⁶ conidia/ml) for half an hour. The negative control was treated with distilled water. All of the plants were then placed in a greenhouse at 28°C, 60% relative humidity, and a 12-h/12-h light/dark photoperiod. After 7 days, leaves on the inoculated plants turned yellow and withered, and black necrotic areas appeared at the base of the stem. Sections of the stem revealed vascular bundle browning. The negative control plants remained healthy. The pathogen was reisolated from the lesions, and its identity was confirmed morphologically, thereby satisfying Koch’s postulates. To our knowledge, this is first report of F. oxysporum causing Fusarium wilt on imperial chrysanthemum in China.

First Report of Fusarium avenaceum Causing Stem Rot on Polygonatum cyrtonema in Sichuan, China

Polygonatum cyrtonema Hua, which is an important perennial medicinal herb used for the treatment of diabetes and asthma, is planted extensively in China. In May 2018, the plants showed leaf wilt and severe stem rot symptoms. A survey showed 5% of the collected plants were diseased, with pink mold on the stem in the greenhouse and the wild in Sichuan Province. Over time, the necrosis extended around the stem so that the plants collapsed. Morphological and molecular analyses were carried out to confirm the causal agent. Six diseased plant tissues were surface disinfested with 75% ethanol for 30 s, 0.1% HgCl₂ for 1 min, rinsed in sterile water, dried, transferred onto potato dextrose agar (PDA), and incubated at 25°C for 7 days. Morphological characteristics were observed on PDA and synthetic low nutrient agar (SNA) (Garcia-Nunez et al. 2016). The genomic DNA of the isolates was extracted and sequenced to confirm the identity by rDNA internal transcribed spacer (ITS) region, the translation elongation factor 1-alpha (TEF-1α) gene, β-tubulin (TUB2), RNA polymerase II largest-subunit gene (RPB2), and large subunit (LSU) rDNA with primer pairs ITS4/ITS5, EF1/EF2 (O’Donnell et al. 1998), T1/T22 (Wang et al. 2014), fRPB2-5F/fRPB2-7CR (Liu et al. 1999), and LROR/LR5 (Rehner and Samuels 1994; Vilgalys and Hester 1990), respectively. Three isolates from diseased stems with similar morphology were isolated from single spores. Colonies on PDA grew rapidly and generated aerial mycelia with white to orange floccose and carmine to rose pigmentation on the reverse. On SNA medium, macroconidia were falcate, slender, curved with an elongated apical cell, usually three to five septa, measured 22.93 to 57.88 × 2.82 to 5.43 μm (n = 50), and occasionally one to two septa, 18.71 to 26.79 × 2.21 to 4.90 μm (n = 50). Microconidia were usually one-septate, hyaline, ovoid, smooth, and measured 10.09 to 19.39 × 2.22 to 4.28 μm (n = 50). Chlamydospores were not observed. The newly generated sequences (ITS, 561 bp, MK204577; TEF-1α, 673 bp, MK226333; TUB2, 1,261 bp, MK253102; RPB2, 1,125 bp, MK396098; and LSU, 885 bp, MK392034) of the isolate SICAUCC18-0001 indicated that those molecular data had high identity with those of Fusarium avenaceum (Fr.) Sacc. (ITS, KP265354 [100%], KP265365 [100%]; TEF-1α, MG670378 [100%]; TUB2, KY055838 [99%], KP170733 [99%]; RPB2, GQ915486 [99%]; and LSU, MH866319 [100%], MG274300 [100%], MG274299 [100%]) obtained from Solanum tuberosum L., wheat, Beta vulgaris L., and Vitis amurensis Rupr., respectively. The identification was confirmed by multilocus phylogenetic analysis. Based on morphological features and phylogenetic analysis, this fungus was identified as F. avenaceum (Garcia-Nunez et al. 2016). To complete Koch’s postulates, a pathogenicity test of the F. avenaceum was performed on sterile stems of P. cyrtonema. Ten stems wounded with a sterile toothpick were smeared with 20 μl of conidial suspension (1 × 10⁶ conidia/ml). Control plants were treated with sterile distilled water. Plants were placed in an aseptic incubator at 25°C and 95% relative humidity with 12-h light/dark. Seven days later, the inoculated plants developed symptoms similar to those observed in the field, and the controls were asymptomatic. The pathogens were successfully isolated from symptomatic stems. To our knowledge, this is the first report of F. avenaceum causing stem rot on P. cyrtonema.

Recipes and Tools for Culture of Escherichia coli.

In this article, we provide information about culture media, including minimal liquid media, rich liquid media, solid media, top agar, and stab agar. We also provide descriptions and useful information about tools used with growth media such as inoculating loops, sterile toothpicks, and spreaders. © 2018 by John Wiley & Sons, Inc.

First Report of Pseudomonas syringae pv. syringae Associated with Bacterial Blossom Blast on Apple (Malus pumila) in the United States

In May 2017, dead buds at pink and tight cluster growth stages were observed on young apple trees of cultivar SnapDragon at two orchard sites in Marlboro, NY. Disease symptoms included necrosis of lateral and terminal flower buds that were wilted and drooping. Bark below the dead buds on severely affected trees showed black streaking with or without interspersing white streaks. On the cross-section of branch wood, just below the buds, light brown xylem and glossy water-soaked sections resembling frost damage were observed. After bark shaving, dark xylem tissue was visible along the branch length, in contrast to the light or pale color of the healthy tissue. The incidence of the disease on trees was 3 to 5%, and 0.5% of trees died. There were five near-frost events recorded on 4, 8, 9, 10, and 11 May 2017, leading us to suspect that the observed symptoms were blossom blast caused by the ice-nucleating strains of Pseudomonas syringae. Isolation from the affected tree samples was performed on King’s B (KB) and nutrient sucrose agar (NSA) media. After 3 days at 25°C, most of bacterial isolates formed round, shiny, gray-white colonies producing green fluorescent pigment on KB medium and typical levan-positive colonies on NSA. Eight isolates, inducing a hypersensitive reaction on tobacco plants, were further characterized by conventional bacteriological and molecular tests (Braun-Kiewnick and Sands 2001). The analyzed bacterial isolates were Gram-negative, aerobic, hydrolyzed gelatin and aesculin, but not pectate, and were negative for oxidase and arginine-dihydrolase activity. All strains were ice-nucleation active and utilized sucrose, sorbitol, and inositol as carbon sources, but not tyrosine and tartaric acid. Results of levan, cytochrome c oxidase, potato soft rot, arginine dihydrolase, and tobacco hypersensitive reaction (LOPAT) tests indicated that the isolates belong to P. syringae group Ia, and gelatin hydrolysis, aesculin hydrolysis, tyrosinase activity, and tartrate utilization (GATTa) tests designated them as pathovar (pv.) syringae (Kaluzna et al. 2012). Polymerase chain reaction (PCR) with specific primers B1/B2 (Sorensen et al. 1998) and SyD1/SyD2 (Bultreys and Gheysen 1999) showed that isolates possessed genes responsible for synthesis (syrB) and secretion (syrD) of syringomycin, respectively. Partial sequencing of the 16S rDNA gene of three isolates (GenBank MG788015 to MG788017) showed 99 to 100% identity with sequences of P. syringae pv. syringae previously deposited in the NCBI GenBank database. To complete Koch’s postulates, pathogenicity of the four isolates was confirmed by inoculation of 1-month-old potted apple plantlets grown from seeds collected from mature apple fruit of cultivar SnapDragon. One plant per isolate was inoculated multiple times as follows: using a needleless syringe, two leaves per plant were spot-infiltrated with bacterial suspension (10⁶ CFU/ml); on the same plants, stem was also inoculated by inserting a sterile toothpick previously dipped into a bacterial colony. Sterile distilled water was used as a negative control. Inoculated plantlets were sealed within plastic bags for 48 h and further incubated at 22°C. Inoculation test was repeated in two independent experiments. Necrosis of the leaf and stem tissue developed at the point of inoculation, and its progression was observed daily for 2 weeks. No symptoms developed on the control plants. Bacteria were reisolated from inoculated tissues and confirmed as P. syringae pv. syringae by PCR for syrB and syrD genes. This is the first report of P. syringae pv. syringae causing blossom blast symptoms on developing apple buds in commercial orchards in the United States. Even though it rarely occurs on apples, with most recent frosts significantly lowering apple yields in Michigan (2012) and New York (2016), blossom blast might become a more common problem in U.S. orchards. Previously, P. syringae pv. syringae was isolated from dormant apple buds without and with internal necrosis in the United States (Burr and Katz 1984).

First Report of Bacterial Leaf Streak of Corn Caused by Xanthomonas vasicola pv. vasculorum in Oklahoma

Following reports of a new bacterial disease of corn (Zea mays) caused by Xanthomonas vasicola in the nearby states of Colorado, Kansas, and Nebraska (Korus et al. 2017), irrigated corn fields in Caddo, Garvin, Kingfisher, and Texas Counties of Oklahoma were surveyed for symptoms of bacterial leaf streak in August 2016. Symptoms consisting of elongated necrotic lesions mostly confined within leaf veins, with irregular yellow to orange colored margins, were observed in several fields in Texas County, although disease severity was low (less than 5%). Bacterial streaming was observed from excised leaf sections, and yellow mucoid bacterial colonies were present on nutrient agar cultures obtained by streaking aqueous bacterial suspensions from lesion sections. Isolates were purified and grown on nutrient agar for 3 days, and DNA was extracted using a quick method. The quick method consisted of heating an aqueous bacterial suspension (one colony picked with sterile toothpick in 100 μl) for 15 min at 100°C and retaining supernatant following centrifugation at 1,200 rpm for 5 min. The primers Xvv3 that targets membrane protein and Xvv5 that targets putative exported protein of X. vasicola pv. vasculorum (Lang et al. 2017) were used in polymerase chain reaction (PCR) assays to identify the pathogen. Isolates OK-6-1, OK-6-3-1, OK-6-3-2, and OK-8-1 were PCR positive for the expected 207-bp product for Xvv3 and for the 200-bp product for Xvv5 primer pairs, confirming the pathogen in 2 of the 19 fields surveyed. Other bacteria including nonpathogens isolated from the corn leaves and X. euvesicatoria were PCR negative. Pathogenicity tests were performed on 21-day-old plants of corn hybrid 208-71VT2PRIB (two plants per isolate and inoculation method) grown in the greenhouse at 22 to 29°C. Bacterial suspensions (10⁷ CFU/ml of sterile tap water) were sprayed to run-off on plants incubated for 48 h pre- and postinoculation in humidity chambers in the greenhouse at 100% relative humidity (RH), or were infiltrated into leaves of plants left on the greenhouse bench at 50 to 60% RH. Sterile tap water served as the noninoculated control. Typical bacterial leaf streak lesions developed within 7 days postinoculation with the four PCR-positive isolates. Symptoms on spray-inoculated plants were more severe, and the pathogen often killed symptomatic leaves compared with infiltrated leaves, which remained intact 14 days postinoculation. The other bacteria described above and the water control did not cause symptoms. The pathogen was reisolated as described above from a symptomatic leaf representing each isolate and inoculation method. Reisolated bacteria were identified by colony characteristics and the two PCR primers. This is the first report of bacterial leaf streak of corn caused by X. vasicola pv. vasculorum in Oklahoma and expands the current reported geographic range of this new disease in the United States (Korus et al. 2017). The pathogen is synonymous with X. vasicola (Korus et al. 2017) and X. campestris pv. zeae (Qhobela et al. 1990).

Original Article. In vitro evaluation of potato genotypes for resistance against bacterial soft rot (Pectobacterium carotovorum) – a new tool for studying disease resistance

Abstract In vitro screening techniques were used to evaluate 46 genotypes of Iranian potato collection for resistance to bacterial soft rot caused by Pectobacterium carotovorum subsp. carotovorum (Pcc). One month old in vitro rooted potato plantlets were inoculated by two inoculation techniques under in vitro conditions: 1) sterile toothpicks dipped into bacterial suspension and pressed into the crown of plantlets and 2) the freshly cut crown of plantlets were dipped into bacterial suspension of 108 cfu ∙ ml-1 for 10 min. Typical soft rot disease symptoms, including the percentage of wilted leaves were recorded on inoculated plantlets 3, 6, 9, 12 and 15 days post-inoculation. The potato genotypes which were examined responded differently to Pcc and varying levels of resistance were observed. Potato genotype AG showed the highest level of resistance. Results obtained from in vitro screening were then verified by classical tuber slice assay. The verifications were conducted using five representative cultivars: Milva, Ramus, Picaso, Marfona and Agria which responded similarly to both in vitro and classical evaluation systems. Similar results obtained from these tests indicated that the in vitro screening technique developed in this study could provide a simple and rapid whole plant assay in selecting resistant potato genotypes against bacterial soft rot.

Efficacy of mouth rinse in maintaining oral health of patients attending orthodontic clinics

Aim: This study is designed to find out the spirochaete (Treponema denticola ) load in orthodontic patients and evaluate the benefit of mouth rinses containing chlorhexidine and hydrogen peroxide in reducing the bacterial load in the mouth. Objective : Orthodontic procedures are always long with the patients having the appliances placed in the mouth for longer duration. This will compromise the oral hygiene of the patients. Oral prophylaxis is very important to prevent the complications during the treatment period. Physical removal of the debris and plaque is difficult in orthodontic patients. In such case mouth rinses are widely used by many patients. Among the bacteria that colonises the mouth, treponemes dominate because they proliferate rapidly in devitalised tissue with the availability of simple source of nutrient. This study is done to know the presence of spirochetes and to evaluate the effects of mouth rinses containing chlorhexidine and hydrogen peroxide on reducing the spirochetes. Background: Placing orthodontic appliance leads increase in oral microbiota. The spirochetes present in mouth increases due to incomplete removal by normal brushing mechanism henceforth mouth washes provide a safe, effective means of reducing or eliminating the spirochetes found in oral cavity. Materials and methods : Patients undergoing orthodontic treatment are subjected in this study, they were made into three groups, group A:control group (orthodontic patients not taking antibiotics or mouth wash) group B using hydrogen per oxide local smear and group C chlorhexidine mouth wash Samples were collected using sterile tooth picks and transferred to glass slabs and smears are microscopically examined. Result: The variation in the effect of two different disinfectants over the spirochete count in oral flora of orthodontic patients wearing intra oral appliance is evaluated .

First Report of Strawberry Dieback Caused by Lasiodiplodia theobromae.

With a typical Mediterranean climate, Aydin is the third largest strawberry-producing province, responsible for 13% of the overall strawberry production in Turkey. Strawberries (Fragaria × ananassa Duchesne) are mainly grown in raised, plastic-mulched beds under tunnels and soil solarization is the most effectively used management practice to control soil-borne pathogens. During October 2011 and 2012, 2 months after planting, wilting and collapse of plants were observed on commercial strawberry (cv. strawberry Festival) fields in Sultanhisar town of Aydin Province. Eleven percent of the plants were wilted and died. Symptomatic plants exhibited blackened necrotic discoloration of roots and in the cross section of crowns. A fungus was consistently isolated from pieces of infected tissue cut aseptically from the crowns and placed on potato dextrose agar. Fungus produced white colonies and later turned olivaecious black with dense aerial mycelium after 4 to 5 days incubation at 27°C. Dark brown to black pycnidia that formed on 20- to 30-day-old pure cultures under daylight conditions produced abundant conidia that were two-celled, thick-walled, and oval shaped with longitudinal striations. Single spore isolates from 12 samples were obtained and stored for further identification. The average size of 300 conidia was 25.42 ± 2.12 × 12.87 ± 1.08 μm. The morphology of the fungus was similar to Lasiodiplodia theobromae (Pat.) Griff. & Maubl. (syn. Botryodiplodia theobromae Pat.). To confirm the identity of the isolates, the internal transcribed spacer (ITS) region of ribosomal DNA and the elongation factor 1-alpha gene were amplified with the universal ITS1/ITS4 and EF1-688F/EF1-1251R (1) primers, respectively. The amplicons from 12 isolates were commercially sequenced at Macrogen (Korea) and were deposited in GenBank under consecutive accession numbers KF910369 to KF910380 and KJ641536 to KJ641547. Sequence comparison and phylogenetic analysis revealed that all 12 isolates were closely related and belonged to L. theobromae. Pathogenicity tests were performed by the toothpick technique (2) under greenhouse conditions (28°C, 14/10-h day/night, 70% RH) on potted strawberry plants (cv. strawberry Festival). Toothpicks carrying fungal growth taken from 1-week-old corn meal agar cultures of the tested isolates was placed into the basal crown tissue of the plants by piercing about 5 mm depth. Six plants were inoculated for each isolate and six were treated with sterile toothpick for control. All inoculated plants developed wilting and dieback symptoms resembling those of naturally infected plants within 2 to 3 weeks of incubation. All plants inoculated with the tested isolates collapsed after 4 weeks and showed discoloration of internal crown tissue. Control plants did not exhibit any disease symptoms, and crown tissue was symptomless. L. theobromae was successfully re-isolated from lesions of all inoculated plants. L. theobromae has been reported to cause cankers and dieback in a wide range of hosts in tropical and subtropical regions of the world (3). To the best of our knowledge, this is the first report of L. theobromae causing dieback on strawberry plants.

First Report of Xanthomonas campestris pv. campestris as the Causal Agent of Black Rot on Oilseed Rape (Brassica napus) in Serbia.

In September 2010, leaves of oilseed rape (Brassica napus L.) with v-shaped, necrotic lesions on the leaf margins surrounded by yellow halos were collected. Symptoms were observed on the domestic cultivar Slavica (IFVC, Novi Sad) located in the Bačka region, Vojvodina, Serbia, from a 3-ha field. Average disease incidence on 3-month-old plants was 45% (15 to 75%). Diseased leaves were rinsed in sterilized distilled water (SDW) and dried at room temperature for isolations. Leaf sections taken from the margin of necrotic leaf tissue were macerated in SDW and the extract was streaked onto yeast extract-dextrose-calcium carbonate (YDC) agar. Plates were incubated at 28°C for 3 days. Colonies were yellow, translucent, circular, and raised. Ten representative strains tested further were all gram-negative, catalase-positive, and oxidase-negative. The partial 16S rDNA sequence of a representative strain, TUr1, was amplified using primers fD1 and rD1 (2), and determined using the IMGGI SeqService facility in Belgrade. The 1,510-bp 16S rDNA sequence of TUr1 was compared to that of known strains in the NCBI GenBank database, and showed greatest similarity with that of Xanthomonas campestris pv. campestris (Xcc) strains ATCC 33913 and B100 (99% homology). Pathogenicity of 10 strains grown for 48 h on YDC at 28°C was completed using each of three methods: spraying a bacterial suspension (108 cfu/ml) onto the leaf surfaces of oilseed rape plants, stabbing the major veins of each of the first two true leaves with the tip of a sterile toothpick that had been dipped into a colony of the appropriate strain, and immersing cotyledons of the plants into a bacterial suspension (108 cfu/ml). All three tests were performed on 4-week-old oilseed rape plants of the cultivar Slavica. SDW was used for the negative control treatment for each method of inoculation. Reference strain Xcc NCPPB 1144 was used as a positive control treatment. Tests plants (two for each method of inoculation and each bacterial strain or control treatment) were maintained in a greenhouse at 25 ± 1°C and 80% relative humidity by keeping the plants in plastic bags. Two control plants for each of the negative and positive control treatments for each inoculation method were also enclosed in separate plastic bags. The bacterial strains and reference strain caused yellow lesions on inoculated plants that turned necrotic starting about 7 days after inoculation (DAI). The spots coalesced within 21 DAI to form necrotic areas. Plants inoculated with SDW remained symptomless. Reisolations were done onto YDC as described above. Reisolated strains showed the same colony morphology as described above. The bacterial strains grew at 35°C; produced levan from sucrose, hydrogen sulfide, and indole; did not reduce nitrate; hydrolyzed Tween 80; starch, gelatin, and aesculin; did not show tolerance to 0.10 and 0.02% triphenyl-tetrazolium chloride; and produced acid from d-arabinose, arginine, dulcitol, galactose, d-glucose, maltose, mannose, sorbitol, sucrose, and xylose (1). All strains tested by Plate Trapped Antigen-ELISAs (ADGEN Phytodiagnostics, Neogen Europe Ltd., Scotland) reacted with Xcc-specific polyclonal antibodies. Based on these tests, the strains were identified as Xcc. To our knowledge, this is the first report of this pathogen causing black rot of oilseed rape in Serbia.

Molecular Analysis of Streptococcus Mutans associated with Dental Caries in Bangalore Urban Population

Streptococcus mutans has been implicated as cariogenic bacteria. There is an unresolved complexity that exists in this bacterial species. It has been found that there not much literature regarding profiling of this strain of bacteria in Bangalore urban population.Demonstration of Genomic DNA using agarose gel electrophoresis and PCR-RFLP Analysis of streptococcus mutans in our society may help in contributing towards an empirical approach to therapy that might allow more conservative treatment options while treating dental caries in our population. In this report A total of 100 samples were collected using sterile toothpicks namely S1 to S100 with Phosphate Buffer Saline solution PBS as a transport media. From these 100 samples 56 were isolated and identified as positive and used for this study. Single band of high molecular weight DNA was observed in all the isolates and one standard MTCC497. The dex genes of S. mutans clinical isolates were successfully amplified. The bands were analyzed using DGelDAS which showed that the bands sizes were asymp520-bp to 570-bp. The sizes of PCR products were compared with 600-bp molecular weight DNAladder. The amplified products were successfully restricted with HaeIII. All the samples gave bands whose molecular sizes were compared with the 100 bp DNAladder. The band pattern did not show much of heterogeneity.

Macrophomina phaseolina and Its Association with Strawberry Crown Rot in Australia

Macrophomina phaseolina has been associated with serious crown rot losses in Australian strawberries in recent years. The fungus produces microsclerotia on standard media and dark ostiolate pycnidia when grown on sterile strawberry crown tissue and wooden toothpicks. Experiments in a glasshouse to confirm Koch's postulates indicated the pathogenicity of the fungus in two cultivars, Sugarbaby and Treasure. In the field, a range of soil fumigants killed buried microsclerotia, but only a mixture of methyl bromide and chloropicrin (50:50) eradicated the fungus in infected crowns buried in the soil. This work and worldwide reports suggest that this pathogen may become more important in strawberries in the absence of methyl bromide.

First Report of Pseudomonas viridiflava Causing a Bacterial Blight of Artichoke Bract Leaves.

In 2006, a disease was observed on two artichoke (Cynara scolymus L. cv. Lardati) fields in Crete, Greece, covering ~2 ha. Symptoms developed after several days of rainy and windy weather and >70% of capitula were affected, resulting in unmarketable produce. Initial symptoms were water-soaked, dark green spots on bracts with many sunken, necrotic, often elongated lesions, each with a brown-black center and surrounded by a water-soaked halo with a dark red-brown margin. Symptoms were more severe on inner bracts. Isolations from symptomatic, surface-disinfected bracts onto King's B agar medium (KB) consistently yielded yellow bacterial colonies that produced a green-blue fluorescent pigment. Ten selected artichoke isolates, all gram-negative, presented the LOPAT profile (- - + - +) and were levan negative, oxidase negative, potato rot positive, arginine dihydrolase negative, and showed tobacco hypersensitive reaction. All isolates used L-arabinose, D(-)-tartrate, and L-lactate, but not sucrose, L(+)-tartrate, or trigonelline. Results were identical to those obtained with the reference strain of Pseudomonas viridiflava NCPPB 1249 (3), and strains PV3005 and PV3006 from eggplant (1). Based on these biochemical tests, 10 isolates were identified as P. viridiflava group II members of the LOPAT determinative scheme of Lelliott (1,2). Two artichoke isolates (PV608 and PV609) were selected for molecular characterization. The identity and phylogenetic analysis were determined by multilocus sequence typing with the gyrB, rpoD, and rpoB genes (PV608 Accession Nos. JN383375, JN383363, and JQ267546; PV609 Accession Nos. JN383376, JN383364, and JQ267547). BLAST searches showed highest nucleotide sequence identity (96%) with GenBank sequences of P. viridiflava reference strains NCPPB 963 and CFBP 2107. Pathogenicity of 10 artichoke isolates and reference strains was tested twice on detached capitulum bracts of artichoke cv. Lardati, as well as 4-week-old tomato plants of cv. ACE, and Chrysanthemum indicum cv. Reagan plants. Each isolate was inoculated onto 10 bracts by placing 15 μl of bacterial suspension (5 × 106 CFU/ml) of a 48-h culture in KB broth on the surface of the bract, and pricking the bract through the drop of bacterial suspension with a sterile needle. Each isolate was also inoculated onto five tomato and five chrysanthemum plants by dipping a sterile toothpick in the appropriate bacterial culture and pricking the surface of the stem. Ten control plants were inoculated similarly with sterile, distilled water. Inoculated bracts and plants were kept in boxes lined with moist filter paper at 25 to 30°C and 80 to 100% relative humidity. Lesions developed on detached bracts within 72 h and were similar to those observed on the naturally infected plants. On tomato and chrysanthemum plants, pith necrosis and wilting symptoms were induced within 1 week of inoculation. Symptoms were not observed on control bracts and plants. Bacterial colonies were reisolated from bract lesions and stems with pith necrosis, but not from control plants, and the reisolates had the same LOPAT profile as the original isolates of P. viridiflava, thus fulfilling Koch's postulates. To our knowledge, this is the first report in the world of P. viridiflava causing a disease of artichoke bracts.

First Report of Bacterial Wilt Caused by Erwinia tracheiphila on Pumpkin and Watermelon in New Mexico.

Pumpkin (Cucurbita pepo L., cv. Magic Lantern) and watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai, cvs. Millionaire and Sangrea) plants with wilting leaves and collapse of entire vines were observed during the 2005 and 2006 growing seasons in several fields in southwestern New Mexico (Luna and Hidalgo counties) with an incidence ranging from 7 to 25% and less than 1% in pumpkin and watermelon fields, respectively. Sticky, hyaline strands were visible when vines were cut transversally, indicative of bacterial wilt caused by Erwinia tracheiphila (2). In the pumpkin fields, 12-spotted cucumber beetles, vector insects of E. tracheiphila, were found on plants at the first-true-leaf stage, which were treated with dimethoate. At the 4- to 5-leaf stage, 5 to 10% of the plants were wilted and were removed by hand. Less than 1% of the plants showed symptoms prior to bloom, when a high population of beetles was observed, and the fields were treated with thiamethoxam. To isolate the causal agent of the wilt symptoms, six, 1-cm vine segments and three to five beetles were surface sterilized in 1% NaOCl for 2 min, rinsed and macerated in sterile distilled water, and plated onto potato dextrose agar, nutrient agar, and King's medium B. After incubation at 25°C, bacterial colonies emerged on all media and were grayish white-to-cream, circular, smooth, and glittering. Isolated bacteria were gram negative, did not grow at 39°C, produced hydrogen sulfide gas from hydrolysis of cysteine, and did not hydrolyze litmus milk and starch. With Ready-To-Go PCR beads and 16S rDNA-based primers ET1/ET2 (1), a 700-bp product was obtained from each of two isolates, consistent with previously reported data for E. tracheiphila (1,3). For the pathogenicity tests, 10 seedlings of pumpkin cv. Magic Lantern and watermelon cv. Millionaire were inoculated with each isolate in the greenhouse at the second fully expanded leaf stage using two methods. In the first method, stems were injected with bacterial suspension (106 CFU/ml) using a hypodermic needle. In the second method, a dab of bacterial colonies was taken with a sterile toothpick to stab the cotyledonary axils. Control seedlings were stem injected with distilled water or stabbed with a sterile toothpick. The experiments were conducted four times. Inoculated plants were placed in a humid chamber at 23 to 25°C. Plant wilting was observed within 4 days when stab inoculated with toothpicks and within 7 to 10 days when stem injected with bacterial suspension. Bacterial colonies recovered from inoculated plants were identical to those recovered from field infected plants. To our knowledge, this is the first report of bacterial wilt on pumpkin and watermelon in New Mexico.

NEW HOSTS OF MACROPHOMINA PHASEOLINA IN IRAN

The host range and geographical distribution of Macrophomina phaseolina, the causal agent of charcoal rot or ashy stem blight, was studied in different provinces of Iran during 2006-2008. Plants with signs and symptoms of charcoal rot showed leaf yellowing, blackening of the stem and root rot. Numerous black microsclerotia were observed on the rotted tissue. To isolate the pathogen, infected tissues were surface-disinfected and placed on potato dextrose agar plates Pure cultures were obtained from hyphal tips and maintained on sterile toothpicks at room temperature (Edmunds, 1964). The mycelium that was initially hyaline, turned grey with time and after 4 to 5 days incubation, produced minute, black, round to oblong or irregularly shaped microsclerotia with mycelial attachment. In 10-day-old cultures, microsclerotia ranged in size from 62 to 189×52 to 149 (116×93) μm. All the isolates were identified at the species level based on the amplification of the ITS region with species-specific primers (Babu et al., 2007). Fungal attacks were observed on marigold (Tagetes erecta), cantaloupe (Cucumis melo var. cantaloupensis), cumin (Cuminum cyminum), hemp (Cannabis sativa), mung bean (Vigna radiata), okra (Abelmoschus esculentus), tomato (Lycopersicon esculentum), turnip (Brassica rapa), and watermelon (Citrullus lanatus), which are reported as new hosts for M. phaseolina in Iran. Pathogenicity tests, repeated twice, were conducted in a greenhouse on each of the above host as described by Abawi and Pastor-Corales (1990). The outcome of inoculation assays was assessed after 2-6 months according to the host. Inoculated plants produced typical symptoms on the leaves, stem and roots, and the fungus was consistently re-isolated from them.