Water Agar Research Articles

First Report of Fusarium Wilt of Blackberry Caused by Fusarium oxysporum f. sp. mori in North Carolina

HomePlant DiseaseVol. 104, No. 3First Report of Fusarium Wilt of Blackberry Caused by Fusarium oxysporum f. sp. mori in North Carolina PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Fusarium Wilt of Blackberry Caused by Fusarium oxysporum f. sp. mori in North CarolinaA. M. Pastrana, W. O. Cline, T. W. Wong, D. C. Watson, J. Mercier, K. Ivors, J. C. Broome, L. M. Quesada-Ocampo, and T. R. GordonA. M. Pastranahttp://orcid.org/0000-0001-6540-4573University of California, Davis, CA 95616Search for more papers by this author, W. O. ClineNorth Carolina State University, Raleigh, NC 27695Search for more papers by this author, T. W. WongNorth Carolina State University, Raleigh, NC 27695Search for more papers by this author, D. C. WatsonUniversity of California, Davis, CA 95616Search for more papers by this author, J. MercierDriscoll’s Inc., Watsonville, CA 95076Search for more papers by this author, K. Ivorshttp://orcid.org/0000-0002-0930-3214Driscoll’s Inc., Watsonville, CA 95076Search for more papers by this author, J. C. BroomeDriscoll’s Inc., Watsonville, CA 95076Search for more papers by this author, L. M. Quesada-Ocampohttp://orcid.org/0000-0002-9072-7531North Carolina State University, Raleigh, NC 27695Search for more papers by this author, and T. R. Gordon†Corresponding author: T. R. Gordon; E-mail Address: trgordon@ucdavis.eduhttp://orcid.org/0000-0002-1715-6509University of California, Davis, CA 95616Search for more papers by this author AffiliationsAuthors and Affiliations A. M. Pastrana1 W. O. Cline2 T. W. Wong2 D. C. Watson1 J. Mercier3 K. Ivors3 J. C. Broome3 L. M. Quesada-Ocampo2 T. R. Gordon1 † 1University of California, Davis, CA 95616 2North Carolina State University, Raleigh, NC 27695 3Driscoll’s Inc., Watsonville, CA 95076 Published Online:22 Jan 2020https://doi.org/10.1094/PDIS-09-19-1980-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Beginning in 2015, wilting blackberry plants (Rubus L. subgenus Rubus) were observed in commercial plantings of cultivars BD467.1, BJ110.2, and Prime-Ark 45 in Pender County, North Carolina. Symptoms included chlorotic, wilted, or dried leaves on the entire cane. A dark, necrotic lesion on one side of the cane extending along the entire length of the cane was often visible. Complete or partial plant collapse was also observed, and up to 15% disease incidence occurred during fall 2018. Symptomatic blackberry plants of cultivar Prime-Ark 45 at one location in Pender County were sent to the North Carolina State University Plant Disease and Insect Clinic. To recover fungi from plants, infected tissues were surface sterilized in 1% NaOCl for 1 min. The tissue was rinsed in sterile distilled water, cut into small pieces, and placed on acidified potato dextrose agar (APDA). Two isolates, each from a different plant, were transferred from APDA to Nash and Snyder medium (Thies and Levi 2007), which is selective for Fusarium spp. Single strains (GL1963 and GL1964) were obtained by excising hyphal tips from recently germinated spores. Both isolates produced microconidia in false heads on short monophialides and macroconidia with elongated, bent apical cells and notched basal cells on carnation leaf agar, consistent with identification as Fusarium oxysporum Schlechtendahl emend. Snyder & Hansen (Leslie and Summerell 2006). Primer pairs iNL11/CNSa and iCNS11/NLa were used to amplify the intergenic spacer (O’Donnell et al. 2009). On the basis of a comparison of 2,281 bp, both isolates had identical sequences (GenBank accessions MN013360 and MN013361), which revealed a 100% match with sequences of F. oxysporum accessions in GenBank (AY527725.1) and Fusarium-ID (FD_00847_IGS) databases. A comparison with previously described blackberry isolates showed a 100% match with F. oxysporum f. sp. mori isolates from California (GenBank accessions KY515229 and KY515230). Complementary nit phenotypes obtained for each isolate, as described by Correll et al. (1987), were not compatible with F. oxysporum f. sp. mori strains previously identified in California. Both isolates from North Carolina were tested for pathogenicity to blackberry cultivar BJ110.2. For each isolate, five plants (approximately 8 months old) were inoculated by immersing roots for 10 min in a suspension of 5 × 106 spores/ml, which was obtained from cultures grown on PDA as described by Schmale and Gordon (2003). Thereafter, inoculated plants were placed in pots filled with Sunshine mix number 1 (SunGro Horticulture Canada). Each isolate was included in two independent pathogenicity tests, each of which included isolate GL1804 as the positive control (Pastrana et al. 2017). In each trial, five plants were immersed in sterile 0.1% water agar instead of a spore suspension to serve as negative controls. Inoculated and control plants were arranged in randomly assigned positions in a greenhouse with temperatures ranging from 15 to 30°C and relative humidity between 15 and 75%. By 6 to 7 weeks after inoculation, all inoculated plants were severely diseased or dead, and negative control plants remained healthy. F. oxysporum was recovered from stems and petioles of all symptomatic plants. This disease was previously reported in California and Mexico (Gordon et al. 2016; Hernandez-Cruz et al. 2015).The author(s) declare no conflict of interest.

Colonization of Metarhizium anisopliae on the surface of pine tree logs: A promising biocontrol strategy for the Japanese pine sawyer, Monochamus alternatus

We investigated the colonization potential of five Metarhizium anisopliae isolates on pine tree surfaces under laboratory conditions, determined the influence of the pine bark extract on fungal growth and evaluated the insecticidal activity following colonization on the Japanese pine sawyer. Finally, the effect of colonization on adults pine sawyer was evaluated using the top three performing isolates (JEF-197, JEF-271 and JEF-279) under laboratory and field conditions. As a result, isolate JEF-197 showed the highest conidial production on the pine surfaces, and five isolates, including JEF-197, showed higher hyphal growth on autoclaved pine bark extract agar, compared to a water agar. Pine bark treated with the isolates showed 40–70 % mortality of adults pine sawyer. Under mimicked overwintering conditions, in the JEF-197 treatment group, 40 % of the inserted larvae became adults and all were dead after 59 d. In a field test, colonized isolate JEF-197 also showed 37 % insecticidal activity against emerged adults from the pine logs as overwintering sites. This work suggests that M. anisopliae isolate JEF-197 possibly colonized the pine surface and application of a conidial suspension on the pine logs as overwintering sites could be an effective strategy to control the pine sawyer.

PRODUCTION OF BIOPLASTIC FROM POTATO STARCH

Due to their diverse properties, plastic materials are used in numerous sectors. It is possible to produce different articles and plastic objects with reduced costs, being more accessible to the population. Conventional plastics are obtained from petroleum-derived raw materials, a non-renewable resource in which their extraction and refining process cause major environmental impacts. The production of plastic reaches a level of approximately one hundred and forty million tons per year, and the disposal of these materials is increasing, generating a high rate of waste and leading to an increase of pollution since the decomposition of these materials lasts about five hundred years old. Conventional plastics can be replaced by bioplastics, a material obtained from renewable raw materials such as potatoes, cassava, maize, and which, when disposed of under favorable conditions, decomposes faster, as during its degradation process at least one step occurs. Through the metabolism of organisms present in the environment. Starch has been widely used in the production of biodegradable packaging, so the objective of this work was to produce a biodegradable bioplastic from the potato starch. Potato starch, glycerin, hydrogen peroxide, distilled water, and commercial agar were used to produce the bioplastic. Bench-scale bioplastics had good organoleptic characteristics, similar in appearance to a conventional plastic obtained from petroleum. The thickness, moisture content, and solubility of the bioplastics were analyzed, as well as their fruit preservation capacity. The samples produced were rigid and with good resistance.

The Influence of Some Sources of Nitrogen on the Growth and Development of the Phomopsis Incarcerata Pathogen (SACC.) Hohnel

Phomopsis incarcerata, known as the pathogen which caused the dieback of Rosa branches was detected in many orchards in Romania. Our investigations have approached a series of bio-ecological aspects of this pathogen: isolation, purification and obtaining the pathogen; determination and identification of the pathogen; establishing in vitro parameters of fungal development (nitrogen source). The isolate used in this study was obtained from Rosa spp. branches and was cultivated on three culture media: potato dextrose agar (PDA), malt extract agar and water agar which included five amino-acids: cysteine, glycine, beta-alanine, leucine and tryptophan. Leucine and glycine were favorable for the dynamics of the fungus.When the fungus grew on water agar, the sporulation was completely inhibited. The optimum pH values for the growth and creation of the Phomopsis incarcerata are in the range of 4.4-7, so weak to neutral acid.

The Influence of Some Sources of Nitrogen on the Growth and Development of the Phomopsis Incarcerata Pathogen (SACC.) Hohnel

Phomopsis incarcerata, known as the pathogen which caused the dieback of Rosa branches was detected in many orchards in Romania. Our investigations have approached a series of bio-ecological aspects of this pathogen: isolation, purification and obtaining the pathogen; determination and identification of the pathogen; establishing in vitro parameters of fungal development (nitrogen source). The isolate used in this study was obtained from Rosa spp. branches and was cultivated on three culture media: potato dextrose agar (PDA), malt extract agar and water agar which included five amino-acids: cysteine, glycine, beta-alanine, leucine and tryptophan. Leucine and glycine were favorable for the dynamics of the fungus.When the fungus grew on water agar, the sporulation was completely inhibited. The optimum pH values for the growth and creation of the Phomopsis incarcerata are in the range of 4.4-7, so weak to neutral acid. Keywords: Phomopsis, nitrogen sources, Rosa, pathogen

First Report of Cercospora Leaf Spot Caused by Cercospora chenopodii on Spinacia oleracea in the U.S.A.

In November 2017, leaf spots were observed in a fresh market spinach crop of cultivar SV2157VB in New Jersey at ∼20% incidence but not in the adjacent crop of Unipack-151. Pale tan lesions (3 to 8 mm in diameter) had gray sporulation at the centers. Symptomatic leaf pieces were surface-sterilized in 0.6% NaOCl for 60 s, triple-rinsed in sterilized water, dried, and plated onto PDA and water agar (each with 100 µg of chloramphenicol/ml). Seven isolates all produced hyaline, straight to slightly curved conidia, mostly two- to three-septate, 20.0 to 82.0 µm (45.2 ± 11.3 µm, mean ± standard deviation) × 6.0 to 11.0 µm (8.1 ± 1.0 µm) (n = 312), with no significant differences among isolates. Spore characteristics matched those of Cercospora spp. (Świderska-Burek 2015). The consensus sequence of the calmodulin gene (cmdA) amplified from all seven isolates using primers CALDF1 and CALDR (Lawrence et al. 2013) (GenBank accession no. MN422444) was 99.78% identical to that of Cercospora cf. chenopodii (JX142839.1), which is not differentiated from C. chenopodii by this gene (Groenewald et al. 2013). Actin (actA) primers ACT-512F and ACT-783R generated an identical sequence for the isolates (MN422442) that was 99.55% identical to that of C. chenopodii (JX143081.1) (Carbone and Kohn 1999). The translation elongation factor 1-α (TEF1-α) sequence of the isolates (MN422445) had 99.45% identity to the sequence of a voucher specimen of C. chenopodii (JX143327.1). C. beticola-specific primers CbCALF and CbCALR did not amplify DNA of these isolates, only DNA of control isolates of C. beticola (Knight et al. 2019). Based on a phylogenetic tree inferred from concatenated actA, cmdA, ITS rDNA, and TEF1-α sequences, the isolates clustered with the voucher strain C. chenopodii CBS 132620. Six isolates were each tested for pathogenicity on four 33-day-old plants of each of spinach cultivars Mandolin, SV2157VB, Unipack-151, and Viroflay; sugar beet cultivar KDH4-9 (USDA National Plant Germplasm System PI 683513, homozygous susceptible to C. beticola), red beet cultivars Red Ace and Ruby Queen, and Swiss chard cultivar Silverado. Plants were enclosed in plastic bags for 24 h, atomized with a spore suspension (1 × 10⁴ conidia/ml) prepared from clarified V8 agar plates of each isolate, and returned to the bags for 24 h. Four non-inoculated control plants of each cultivar were atomized with water as control treatments. Beet and chard plants were placed in a greenhouse and spinach plants in a growth chamber with 10 h lighting/day to avoid bolting under 16-h summer days in Washington. Symptoms had not developed after 7 days, at which time spinach plants were moved to the greenhouse. By 9 days, leaf spots resembling those on the original leaves were observed on Mandolin and SV2157VB. By 14 days, there were leaf spots on all four spinach cultivars. Symptoms were most severe on SV2157VB (18.3 ± 2.0% mean ± standard error of leaf area), followed by Mandolin and Viroflay (8.8 ± 1.6%), and then Unipack-151 (2 ± 0.5%). Symptoms did not develop on non-inoculated spinach or inoculated and non-inoculated sugar beet, table beet, and Swiss chard. Cercospora could only be reisolated from symptomatic spinach plants, with the isolates confirmed as C. chenopodii based on actA sequences. Although C. beticola has been reported to cause Cercospora leaf spot of spinach (Knight et al. 2018), this is the first report of C. chenopodii causing a leaf spot of spinach in the United States. Research is needed to evaluate impacts of this species on additional spinach cultivars and management options.

First Report of Leaf Blight Caused by Arthrinium arundinis on Tea Plants in China

Tea (Camellia sinensis [L.] Kuntze) is one of the most important economic crops in China. During a survey in July 2018, symptoms of brown leaf patches (3 to 4 cm diameter) with irregular margins were observed on tea leaves of Longjing43 cultivar in Huoshan tea farm, Anhui province, China. The disease incidence reached 18 to 20% in the most severely affected tea gardens. Lesions first appeared as gray-brown dots, subsequently expanding to irregular dark brown, and then ended with large brown patches on the tea leaves. The infected tea leaf samples were collected from the tea plants, and the pathogenic fungus isolation was performed according to the method described by Thangaraj et al. (2018). Briefly, the tea leaf samples washed with tap water followed by distilled water. The margins of infected leaves were cut into small pieces (4 to 5 mm²) and surface sterilized with 0.1% HgCl₂ for 1 min followed by 70% ethanol for 30 s and rinsed three times in sterile distilled water. Then, these aseptic tissues were blotted dry on sterilized filter paper, transferred onto 2% water agar, and incubated in the dark at 28°C until fungal hyphae started to grow. Single hyphal tips were transferred onto potato dextrose agar (PDA) plates and incubated for 7 days at 28°C for morphological observations. A total of five isolates were obtained and identified as the same fungus on the basis of phenotypic characteristics. The colonial characters of the isolated fungus on PDA were dirty white and moderate aerial mycelium with dark colored patches. Hyphae were smooth, hyaline, branched, and septate. Conidiophores were reduced to conidiogenous cells, size ranged from 4.2 to 11.0 × 2.7 to 5.7 μm (length and width, respectively) (average 7.4 × 3.8 μm; n = 30), pale brown, smooth, and ampulliform. Conidia were 4.5 to 12.2 × 3.4 to 9.4 μm (average 6.9 × 5.0 μm; n = 50), single celled, brown, smooth, anomalous shape (globose to ellipsoid shape), mostly globose in surface view and lenticular in side view with an equatorial slit. Based on morphological characteristics, the isolated plant pathogenic fungus, named ZZZ3, was identified as Arthrinium spp. (Corda) Dyko & B. Sutton (Crous and Groenewald 2013). To identify the isolate to the species level, genomic DNA was extracted (Fungal DNA extraction kit, BIOMIGA, China) from mycelial mats. The internal transcribed spacer region (ITS1/ITS4) (White et al. 1990) and D1/D2 region of 28S rDNA (LR0R/TW13) genes (Hamayun et al. 2009) were amplified. The obtained ITS (539 bp) and 28S rDNA (585 bp) (GenBank accession nos.: ITS, MN187298; 28S rDNA, MN187305) sequence BLAST analysis showed 100 and 99% similarity with Arthrinium arundinis (ITS, KF144883; 28S rDNA, KF144929), respectively. Based on these morphological and genetic characteristics, the fungus was identified as A. arundinis. To confirm its pathogenicity, ten 2-year-old tea plants were selected, and the adaxial surface of matured leaves was injured (two needle punctures per leaf, using a sterile needle). The conidial suspension (10⁶/ml) was spread over the wounds, and sporeless sterile water was spread on wounded control leaves. After inoculation, each plant was covered by a plastic bag containing moist cotton wool for 10 days at 28°C to maintain high relative humidity (85 to 90%). At 14 days following inoculation, lesions appeared on the wounded leaves, and the symptoms were similar to those described above, whereas the control and unwounded leaves remained healthy. The experiment was performed thrice, and the same fungus was reisolated from the infected leaves, showing similar morphological characteristics and molecular traits. Wang et al. (2018) identified eight new Arthrinium spp. from different host plants in China, of which A. xenocordella and A. camelliae-sinensis were isolated from tea plants. To our knowledge, this is the first report of A. arundinis causing tea leaf blight in China. The establishment of the disease in tea plants may require the development of effective control strategies to minimize its impact on the production of tea.

Viability and pathogenicity of Rhynchosporium secalis after long-term storage

Long-term storage of Rhynchosporium secalis cultures is a challenge for any lab managing a working collection of isolates. In this work, the viability and pathogenicity of R. secalis stock cultures were tested after four years of storage at −20 °C in different concentrations of glycerol. Germinability were measured after each storage by collecting spores by coverslips and placing them on water agar in closed Petri dishes at 20–22 °C in the dark and allowed to germinate for 24 h. Additionally, at the end of each storage treatment, conidia were collected by coverslips from sporulated leaf lesions of symptomatic barley leaves and placed under similar conditions as non-stored controls. Cultures of all stored isolates were viable with a spore germination rate of 72.28% (Rs22) after four years of storage at −20 °C in 60% glycerol. Low viability and contamination were observed when spores were stored in sterile distilled water and in Lima bean agar. All isolates continued to infect barley leaves after 4 years of storage. However, the pathogenicity was significantly (P <0.05) reduced in isolates stored in glycerol as compared with controls. This work helps to preserve R. secalis for a long term period at −20 °C without any contamination; therefore, due to the low costs our results could be applicable for laboratories that have limited resources.

Antimicrobial Activity of Endophytic Fungi Isolated from Eryngium foetidum, an Ethnomedicinal Plant of Assam

Eyngium foetidum L. is a medicinal plant widely used by ethnic tribal communities of Assam as an alternative source of medicine for the treatment of various diseases. The present investigation was undertaken with an aim to isolate, identify and assess the antimicrobial activity of endophytic fungi associated with the healthy leaf tissues of E. foetidum. The endophytic fungi were isolated three different media, namely, Potato Dextrose Agar (PDA), Malt Extract Agar (MEA) and Water Agar (WA) media and from three different sites. Altogether, 84 endophytic fungal isolates were isolated from 150 segments of leaf tissues. Dominant endophytes were found to be fungi belonging to the genus Colletotrichum, followed by non-sporulating members grouped under mycelia sterilia. Other fungal genera that were isolated as endophytes were Scopulariopsis, Cladosporium, Stemphylium, Penicilllium and Alternaria. The endophytic fungi thus isolated were studied for antimicrobial activity against some clinically significant human pathogenic test organisms. Ethyl acetate extracts of all endophytes exhibited antimicrobial activity by inhibiting at least one out of all the test pathogens. Amongst the isolated fungi, extracts obtained from three endophytes showed wide-spectrum activity against all the test organisms. The fungal endophytes were identified as Scopulariopsis sp., Penicillium sp. and a sterile isolate morphotype strain EF6. The study indicated that E. foetidum harbours a wide range of endophytes with antimicrobial properties and further detailed investigation of the compound present in them would lead to their potential therapeutic applications as a new source of medicine.

First Report of a Leaf Spot Disease Caused by Alternaria protenta on the Datura stramonium in Serbia

Datura stramonium L. (jimsonweed) is an invasive and competitive annual weed and a potential reservoir host of pests and pathogens in agricultural fields. During a survey of potato growing fields in the municipality of Maglic, Vojvodina Province, typical Alternaria symptoms on leaves of D. stramonium were observed among the potato plants in September 2016. The incidence of diseased plants ranged from 12 to 70%. The symptoms started as small, round, light brown spots with chlorotic haloes, which gradually enlarged to 0.3 to 2 cm in diameter, becoming irregular and sometimes having a dark brown border. Isolates were obtained by placing small pieces of surface-sterilized infected tissues (immersed in 1% sodium hypochlorite for 1 min and rinsed with sterilized water) on V8 medium for 7 days at 23°C with a 12-h photoperiod. The five isolated colonies were circular, smooth, gray to brownish black, with concentric zones of very intensive sporulation. Conidiophores were branched, solitary or in small groups, brown to olivaceous brown, ranging from 125 to 230 µm. Mature conidia were olivaceous brown, long ovoid or long ellipsoid, 102 to 113 µm long and 16 to 29 µm wide, with five to 12 transverse septa and zero to three longisepta. Beak dimensions were 62 to 98 µm long and 6 µm wide at the base apex to 2 µm on the top of the beak. Two-beak conidia were noticed with a frequency of 28%. Based on morphological characteristics, all isolates were identified as Alternaria protenta Sorauer (Simmons 2007). To further identify the pathogen, PCR was used with the A. solani-specific primer set OAsF7 and OAsR6 (Gannibal et al. 2014). A single unique band of 164 bp was amplified for all five isolates. To confirm the pathogen’s identity, PCR was carried out with isolate JB 46-1 using primers ITS1/ITS4 (White et al. 1990), gpd1/gpd2 (Berbee et al. 1999), and RPB2-5F2/fRPB2-7cR (Woudenberg et al. 2013). The ITS, GAPDH, and RPB2 sequences were deposited in GenBank under accession numbers KY913819, KY913818, and MH454660, respectively, and BLAST analyses showed 100% nucleotide sequence identity with the corresponding gene regions of A. protenta strain CBS 116651 (KC584217, KC584139, and KC584430). A pathogenicity test was carried out with isolates JB 46-1 to JB 46-5 on 20 5-week-old healthy jimsonweed plants grown in a greenhouse at 23 ± 2°C. Inoculation was performed by spraying a conidial suspension (10⁶ spores/ml) prepared from 10-day-old cultures grown on V8 agar plates at 23°C with a 12-h photoperiod. Plants used as controls were sprayed with sterilized distilled water in a similar manner. Inoculated plants were kept in a moist chamber for 2 days with 90 to 100% relative humidity. Leaf spot symptoms, brown lesions with chlorotic halos, similar to those previously observed in naturally infected plants, appeared 5 days postinoculation for all inoculated plants. No symptoms were observed on the inoculated control plants. To fulfill Koch’s postulates, lesions were excised, surface sterilized, and plated on water agar, and the fungus emanating from the infected tissues was identified as previously described. To our knowledge, this is the first report of A. protenta causing leaf spot on D. stramonium in Serbia. According to previous reports, A. protenta can also cause leaf spot disease on potato (Ayad et al. 2017; Landschoot et al. 2017; Woudenberg et al. 2014). The fact that jimsonweed coexists in close proximity with potato plants may have a potentially important economic impact on cultivation of solanaceous crops if left uncontrolled and unmonitored. Therefore, constant management practices are required to control the threat that D. stramonium weed and its diseases might present.

Viability assessment of Bifidobacterium longum ATCC 15707 on non-dairy foods using quantitative fluorescence microscopy

This study demonstrates an effective technique for separating and purifying viable bacteria from samples that interfere with viability staining. The viability of Bifidobacterium longum ATCC 15707 was assessed using Percoll Buoyant Density Gradient Centrifugation (PBDC) to separate bacteria from complex non-dairy food matrices and Quantitative Fluorescence Microscopy (QFM) to determine individual cells using LIVE/DEAD BacLight bacterial viability staining. Water agar (3%) was used to retain cells of B. longum and offered a lower fluorescence background with BacLight viability staining, compared with fixation on polycarbonate (PC) black membrane. The effect of drying temperatures and non-dairy foods on viability of B. longum was assessed. B. longum coated on oat, peanut or raisin was separated by filtration, low- and high-speed centrifugation, flotation and sedimentation buoyant density centrifugation. Purified cells were subsequently deposited on water agar for rehydration followed by LIVE/DEAD BacLight viability staining and enumeration. Conventional plate counting was also conducted to compare viability results. Finally, this method was applied to assess cell membrane damages of B. longum incorporated onto non-dairy foods during 24 h drying. Furthermore, viability assessment of B. longum coated onto oat, peanut, or raisin was much lower by plate counting compared to viability staining. Drying appeared to have a greater impact when viability was assessed by plate counting compared to viability staining. ImportanceEnumeration of viable beneficial bacteria from function foods presents a significant bottleneck for product development and quality control. Interference with microscopic and/or fluorescent techniques by ingredients, time required to incubate plated microbes, and the transient nature of the colony forming unit make rapid assessment of viable bacteria difficult. Viability assessment of Bifidobacterium longum ATCC 15707 by Percoll Buoyant Density Gradient Centrifugation with LIVE/DEAD BacLight viability staining on water agar (3%) was in agreement with serial dilution enumeration. Without the need for incubation viability assessment by staining provided a more rapid means to assess the impact of drying on the viability of B. longum coated onto oat, peanut or raisin.

Mutu Bakteriologis Pindang Tongkol (Euthynnus affinis) yang Berasal dari Beberapa Pasar di Yogyakarta

Food poisoning cases need to be examinated intensively. There have been five poisoning cases reported between 2003-2004 in Sleman area. Three of them were caused by contaminated tuna fish. The objective of this research was to determine bacteriological quality of salted tuna fish from several markets in Yogyakarta. Salted tuna fish sample were collected from five markets, namely Beringharjo, Condongcatur, Demangan, Kranggan and Sentul. In each market, four vendors were selected. Bacteriological test were done including total microbe cells utilizing PCA medium, Coliform test utilizing VRBA medium. Identification and isolation of Vibrio parahaemolyticus use pre-enrichment selective medium of Alkaline Petone Water and selective Chromogenic medium agar for Vibrio. Salmonella test utilizing Chromogenic agar continued by utilizing TSI and LIA medium. The result showed that bacteriological quality of salted tuna fish from markets in Yogyakarta did not fulfill the requirements based on SNI 01-2717-1992. It was shown that total microbial count were high, i.e. 2.4 x 105 – 5.27 x 107 CFU/ g. Coliform count ranged from < 2.5 x 101 - > 6.5 x 106 CFU/g. From 20 samples that were brought to analyzed, 60% was positively presumed contaminated by V. parahaemolyticus and 50% was positively presumed contaminated by Salmonella.

Phyllosticta capitalensis Causes Leaf Spot on Tea Plant (Camellia sinensis) in China

<i>Phyllosticta capitalensis</i> Causes Leaf Spot on Tea Plant (<i>Camellia sinensis</i>) in China

Endophytic fungi from Syzygium cumini (L.) Skeels leaves and its potential as antimicrobial agents

One of the harbours of endophytic fungi was medicinal plants. In this study, medicinal plant of Syzygium cumini (L.) Skeels was used to isolate endophytic fungi, especially from the leaves. The surface sterilization was first step before isolation of endophytic fungi. Isolation of endophytic fungi used potato dextrose agar (PDA) media and PDA containing plant extract. The single spores from endophytic fungi were obtained using water agar method. Thirty-seven endophytic fungi have been isolated from S. cumini and the antimicrobial activity was shown by 17 of endophytic fungi isolates. The result shows that endophytic fungi isolated from leaves of S. cumini (L.) Skeels display a good source of natural antimicrobial agents.

First Report of Leaf Spot Caused by Alternaria alternata on Ginseng (Panax ginseng) in China

Ginseng (Panax ginseng) is a perennial herbaceous plant and a precious traditional Chinese medicine. P. ginseng is widely cultivated in Asia, particularly the Changbai Mountain area in China (Wang et al. 2019). Alternaria species can grow under a wide range of temperatures that occur worldwide (Konstantinova et al. 2002). Alternaria panax was reported as the main cause of Alternaria leaf spot of ginseng (Deng et al. 2013); however, a small-spored Alternaria species was isolated from leaf spot symptoms during recent surveys. In our study, leaf spot disease of P. ginseng was observed during 2017 in the Fusong district, Jilin, China. The disease was widespread, with 20 to 30% of the fields affected. Leaves of the affected plants showed brown to black necrotic spots. The fungus was isolated from symptomatic leaves on potato dextrose agar and characterized morphologically under the microscope. Colonies of pure cultures were initially dark brown and then turned black after 7 days. Strains (TCS 15013001 and TCS 15013002) were grown on water agar embedded with surface-sterilized ginseng spot tissues under a 12/12-h light/dark cycle at room temperature. Conidia observed on the lesions were dark brown or olive-brown, ovoid or ellipsoid, short beaked, with three to seven transverse and zero to five longitudinal septa, and 14.9 to 36.5 μm (average 25.8 μm) × 5.8 to 17.9 μm (average 12.5 μm). Based on its morphological characteristics, the pathogen was identified as Alternaria sp. (Simmons 2007). Single-spore cultures were obtained, and their genomic DNA was extracted. The rDNA internal transcribed spacer (ITS, GenBank accession nos. MN394879 and MN394880), endopolygalacturonase (endoPG, MN410909 and MN410910), Alternaria major allergen (Alta1, MN410911 and MN410912), and the portion of the histone 3 (H3, MN410913 and MN410914), elongation factor 1-alpha (EF1-α, MN410915 and MN410916), the RNA polymerase II 2nd largest subunit (RPB2, MN410917 and MN410918), and glyceraldehyde 3-phosphate dehydrogenase (gpd, MN410919 and MN410920) gene were amplified from the two isolates, respectively (Woudenberg et al. 2015). Sequence analysis of the two isolates showed 99 to 100% identity to that of A. alternata (MG182428 for ITS, MG598798 for endoPG, MG598796 for Alt a1, MG182429 for H3, KU738723 for EF1-α, KU738707 for RPB2, and KP851761 for gpd). Pathogenicity tests were conducted in a greenhouse. Sixty healthy 2-year-old P. ginseng seedlings grown in potting medium were tested in a greenhouse at 20/26°C and about 90% relative humidity with natural light. Of the plants, 30 were sprayed with spore suspension (1 × 10⁵ spores/ml) until most of the leaves were wet. Control treatments (30 plants) were treated with distilled water. The two isolates were tested, and all test plants were covered with clear plastic bags for 3 days to increase humidity. Each treatment had three replicate plants. Six days after inoculation, leaf spots similar to the original ones developed at inoculation sites for all isolates, and A. alternata was consistently reisolated from the induced symptoms. The pathogenicity test was performed three times. A. alternata causing leaf spot of American ginseng was reported in China (Hill and Hausbeck 2008). Also, leaf spot of Panax notoginseng caused by A. alternata was reported in China (Yang et al. 2018). To our knowledge, this is the first report of A. alternata on P. ginseng in China.

First Report of Macrophomina phaseolina Causing Dry Root Rot of Chickpea (Cicer arietinum) in Serbia

HomePlant DiseaseVol. 103, No. 10First Report of Macrophomina phaseolina Causing Dry Root Rot of Chickpea (Cicer arietinum) in Serbia PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Macrophomina phaseolina Causing Dry Root Rot of Chickpea (Cicer arietinum) in SerbiaD. Živanov, S. Tančić Živanov, N. Nagl, A. Savić, S. Katanski, and D. MilićD. Živanov†Corresponding author: D. Živanov; E-mail Address: dalibor.zivanov@ifvcns.ns.ac.rshttp://orcid.org/0000-0002-1011-6715Institute of Field and Vegetable Crops, Novi Sad, SerbiaSearch for more papers by this author, S. Tančić Živanovhttp://orcid.org/0000-0001-9556-2732Institute of Field and Vegetable Crops, Novi Sad, SerbiaSearch for more papers by this author, N. NaglInstitute of Field and Vegetable Crops, Novi Sad, SerbiaSearch for more papers by this author, A. SavićInstitute of Field and Vegetable Crops, Novi Sad, SerbiaSearch for more papers by this author, S. KatanskiInstitute of Field and Vegetable Crops, Novi Sad, SerbiaSearch for more papers by this author, and D. MilićInstitute of Field and Vegetable Crops, Novi Sad, SerbiaSearch for more papers by this authorAffiliationsAuthors and Affiliations D. Živanov † S. Tančić Živanov N. Nagl A. Savić S. Katanski D. Milić Institute of Field and Vegetable Crops, Novi Sad, Serbia Published Online:24 Jul 2019https://doi.org/10.1094/PDIS-03-19-0652-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Macrophomina phaseolina (Tassi) Goid has been reported as a severe pathogen of many plants worldwide (Mengistue et al. 2015), as well as in Serbia (Tančić Živanov et al. 2018). However, there has been no formal report of M. phaseolina causing dry root rot of chickpea (Cicer arietinum L.) in Serbia. In June 2018, the first symptoms were observed on affected field plants grouped in patches on approximately 10% of chickpea plants in a 3-acre area at Rimski Šančevi, Serbia. Withering and death of aerial parts and root rot were observed on chickpea plants at the V6 growth stage. Cuttings from symptomatic root tissues of five chickpea plants were surface disinfected with 1% NaOCl solution for 5 min, rinsed three times in sterile distilled water, air dried on sterilized filter paper, and plated on potato dextrose agar (PDA) and water agar amended with 100 mg/liter of streptomycin sulfate (Sigma-Aldrich). After 7 days of incubation at 28°C in the dark, isolates were preliminarily identified according to their morphological characters, as hyaline mycelia that turned from gray to dark with age and produced black pigmentation in agar caused by masses of dark oblong microsclerotia formed 3 days after incubation (Watanabe 2010). A representative isolate (K349) was purified by a hyphal-tip transfer technique for further analyses (Leslie and Summerell 2006). To confirm the morphological identification, the rRNA internal transcribed spacer (ITS) region of the isolate K349 was amplified with universal primers ITS1 and ITS4. The resulting ITS sequence (GenBank no. MK418768.1) was used in a BLAST search of the NCBI nucleotide database and showed 100% identity with M. phaseolina isolates (e.g., GenBank nos. KJ744350.1, KP784426.1, KU831518.1, etc.). Translation elongation factor 1-alpha gene (TEF1-α) was amplified using primers EF1-728 and EF1-986, and the resulting sequence (GenBank no. MK430416) had 97.2 to 99.5% identity with M. phaseolina isolates (e.g., GenBank nos. MH000354.1, MG434668.1, KX400854.1, etc.). After the amplification with primers MpKF1 and MaKR1, specific for M. phaseolina (Babu et al. 2007), a 350-bp PCR fragment was obtained, indicating that the isolate K349 was indeed M. phaseolina. The pathogenicity was confirmed according to Koch’s postulates. Chickpea seeds were surface disinfected with 1% NaOCl, air dried, and planted in 10-cm-diameter pots filled with sterilized potting mixture. In total, 10 plants (one per pot) were maintained in the growth chamber at a constant temperature of 25°C, 14-h photoperiod, with relative humidity 100%. Eight-day-old plants were inoculated with three PDA plugs of 5 mm diameter around the exposed stem base of each plant and covered with sterile soil mixture. PDA plugs without mycelia were used for control plants. After inoculation, plants were incubated under growth chamber conditions at 28°C with 14-h photoperiod and watered regularly. The first symptoms observed were wilting leaves and discoloration around the stem base 16 days after inoculation, whereas the control plants remained symptomless. The pathogen was successfully reisolated from the stem base and confirmed as M. phaseolina. To the best of our knowledge, this is the first report of M. phaseolina causing root rot on chickpea in Serbia.The author(s) declare no conflict of interest.

Life Cycles of Myxogastria Stemonitopsis typhina and Stemonitis fusca on Agar Culture.

Myxogastria is a group of protozoa characterized by cellular uninucleate amoeboflagellates (myxamoebae and flagellated swarm cell), acellular multinucleate plasmodia, and stationary spore‐bearing sporocarps. The Stemonitales is a large order in the Myxogastria and contains approximately 230 species, but only 13 species have their completed life cycles observed so far. Here, we described the life cycles of two species in Stemonitales, Stemonitopsis typhina and Stemonitis fusca by culturing in water agar medium and observing the morphogenesis of their spore germination, plasmodium, and sporocarp development. The spore‐to‐spore life cycles of Ste. typhina and S. fusca were completed in approximately 67 and 12 d, respectively. Both species possessed an aphanoplasmodium. However, the spores of Ste. typhina and S. fusca germinated by the V‐shape split and pore methods, respectively. Unlike S. fusca with an evanescent peridium, Ste. typhina produced a shiny persistent peridium which was continuous with the membrane surrounding its stalk. The information will contribute to a better understanding of their taxonomy and phylogeny.

First Report of Lasiodiplodia pseudotheobromae Causing Canker and Shoot Dieback on Apple in China

In July 2017, apple trees (Malus pumila Mill.) with canker and shoot dieback symptoms on side branches were observed in several young orchards in Yantai, Shandong Province, China. Symptoms included shoot dieback, discoloration of xylem tissues, cankers, and sunken necrotic bark lesions, which surrounded the branches and resulted in the death of the branches. The disease is symptomatically different from canker diseases of apple earlier described. Small pieces (4 to 5 mm) of necrotic tissues from the shoot cankers were disinfected for 1 min in 75% ethanol, rinsed thrice with sterile distilled water, and plated onto potato dextrose agar (PDA) with 0.05% lactic acid. White colonies developed from symptomatic tissues but became dark gray mycelia with age. Pycnidia were produced on sterilized apple twigs on 2% water agar after incubation for 3 weeks at 25°C. Conidia were unicellular, ellipsoidal, initially hyaline, and became dark brown and developed a central septum with age. Conidia measured 21.3 to 31.9 × 12.4 to 16.9 µm (n = 50). On the basis of morphological characteristics, the isolates were identified as Lasiodiplodia sp., a member of the Botryosphaeriaceae family (Alves et al. 2008; Phillips et al. 2013). For further identification, total DNA was extracted from the mycelium of three monoconidial isolates (La1, La2, and La3). The rDNA internal transcribed spacer (ITS), a partial sequence of β-tubulin gene (TUB), and translation elongation factor 1-α gene (TEF1) were amplified and sequenced using primers ITS1/ITS4, Bt2a/Bt2b, and EF1-688F/EF1-1251R, respectively (Alves et al. 2008; Glass and Donaldson 1995; White et al. 1990). The sequences (GenBank accession nos. MK408640 to MK408642 for ITS, MK446722 to MK446724 for TUB, and MH829861 to MH829863 for TEF1) were aligned using BLASTn in GenBank, showing 99 to 100% homology with the sequences of ITS (EF622077), TUB (EU673111), and TEF1 (EF622057) from L. pseudotheobromae isolate CBS 116459 (Alves et al. 2008). The pathogenicity testing of three isolates was conducted using the wound inoculation method both on living apple trees and detached twigs. Thirty 2-year-old twigs were wounded with a scalpel on the phloem tissues and inoculated by placing 5-mm² agar plugs containing 2-day-old cultured hyphae or 10 μl of an inoculum suspension (10⁵ conidia/ml), which were then sealed with Parafilm. The same number of wounded twigs was inoculated with uncolonized PDA plugs or sterile water as controls. After 7 days, necrotic lesions (35 to 60 mm in diameter) developed around the inoculation points, which resembled the symptoms on infected apple shoots in the field. No symptoms were observed on the control twigs, and the pathogenicity test was conducted thrice. The pathogen was reisolated from symptomatic twigs and was reidentified as L. pseudotheobromae on morphological and molecular analysis. Although L. pseudotheobromae was previously reported on several forest and fruit trees (Alves et al. 2008; Awan et al. 2016), to our knowledge, this is the first report of the pathogen causing canker and shoot dieback on apple in China. This report is important for the new research aiming at management of apple canker disease.

Effect of temperature, pH, physical and chemical factors on germination rate of the chlamydospores of the nematophagous fungus Duddingtonia flagrans.

This study mainly investigated the effects of environmental factors on the germination/dormancy, sporulation and resistance of Duddingtonia flagrans chlamydospores. Results showed that the germination temperature of chlamydospores was >10°C and ≤35°C. After the chlamydospores were treated at -20, -40 and -80°C for 12-24 h, they still had the ability to germinate. The chlamydospores germinated at pH 3-13 but did not germinate at pH 1-2 and pH 14. The chlamydospores could tolerate ultraviolet rays for 720 min, but visible light irradiation for 24 h significantly reduced their germination rate. The chlamydospores did not germinate under anaerobic conditions. After the chlamydospores were cultured on water agar (WA) containing 5, 10 and 20% NaCl, their germination rate was significantly inhibited. Once NaCl was removed, the chlamydospores almost completely recovered their germination ability. Among the nine kinds of additives used in the study, 0.3% arginine significantly promoted spore germination (P<0.05) but 1% trehalose and 1% glycerine significantly inhibited spore germination during incubation from 24 h to 48 h (P<0.05). This work indicated that D. flagrans chlamydospores are highly resistant to environmental variations and so could be used for biocontrol of animal parasites.

Heat treatments of dormant scion wood killed the European canker pathogen &lt;i&gt;in planta&lt;/i&gt;, while chemical treatments did not

Neonectria ditissima, the causal agent of European canker, can be present in symptomless scion wood. Sanitation treatments could minimise this risk to nursery trees. In this trial, six heat treatments and five chemical treatments were tested for their effectiveness in removing this pathogen from dormant ‘Royal Gala’ wood. In July 2018, 120 symptomless inoculated shoots (three inoculations/shoot) were harvested and stored at 1oC for 3 months. Bundles of five inoculated shoots (45 cm) were placed in the centre of 24 bundles, each consisting of 25 wood pieces. Heat-treated bundles were submerged in water (45oC for 45 min or 50oC for 15 min), or wrapped in moist cloth, vacuum sealed inside plastic then submerged for 3–6 h at the same temperatures. Chemical-treated bundles were submerged for 16 h at room temperature. Treatments were compared with untreated wood. After surface sterilising, isolation of N. ditissima from inoculated wounds was attempted on apple-sap amended water agar. All wounds from the untreated wood and from the chemical-treated wood yielded the pathogen. However, N. ditissima was not isolated from wounds that had been heat treated. Therefore, heat treatments that do not affect scion wood viability may prove an effective tool to remove European canker from nursery material.