Surface Of Potato Dextrose Agar Research Articles

Duration Effects of Shrimp Paste Storage on ALT of Mold Colonies: Variations by Brand and Pre-Standard Treatment

Shrimp paste is a commonly employed condiment or flavoring in culinary preparations. As a result of the nutrients present in shrimp paste, fungi and other microbial contaminants are able to proliferate and develop on the paste. This study aims as follows: 1) to determine ALT measurements of mold colonies to assess the quality of two brands of pre-treated shrimp paste; and 2) to identify contaminating mold species present in steamed and unsteamed shrimp paste prior to storage. Descriptive quantitative and qualitative research methods were employed within the Biology Department of FMIPA UM's Microbiology Laboratory. A series of three samplings were conducted on treated shrimp paste brands A and B, one week apart between each sampling. A volume of 90 ml of 0.1% peptone water was used to dissolve 10 grams of shrimp paste, resulting in a dilution of 10-1; this process was repeated until the dilution reached 10-5. 0. The surface of PDA (Potato Dextrose Agar) medium was inoculated with 1 ml of suspension from each dilution. The medium was subsequently incubated at a temperature of 25oC for 7x24 hours. The findings of this study indicate the following: 1) At the 28th day of storage, both brands of shrimp paste that underwent treatment with steamed and unsteamed shrimp paste remained of acceptable quality for human consumption. 2) In steamed and unsteamed shrimp paste, thirteen species of mold contaminants were identified: Chrysosporium corda, Cladosporium sphaerospermum, Penicillium frequentans, Penicillium chrysogenum, Rhizoctonia sp1., Aspergillus candidus, Fusarium equeseti, Colletotrichum ti, Moniliella acetobutens, and Rhizoctonia sp3

First report of Alternaria alternata causing leaf spot on Hibiscus mutabilis in China.

Hibiscus mutabilis L. is a deciduous shrub native to China. Because of its ornamental value and ecological value, it has been widely cultivated in many provinces of China (Shang et al. 2020). In October 2021, leaf spot on Cotton rose with about 80% disease incidence was observed in Jinan (116.9408° N, 36.6688° E), Shandong, China. Symptoms first appear on leaves with small dark brown spots surrounded by yellow halos, then become irregular necrotic spots with yellow halos. The diseased leaf samples were packed in paper bags and transferred to the laboratory for isolation. The infected leaves were firstly surface-sterilized for 45 seconds in 75% ethanol, 1 min in 1% sodium hypochlorite, and 1 min in 75% ethanol, then rinsed for 2 min in distilled water and blotted on dry sterile filter paper. Then samples were cut into 5 × 5 mm pieces using a double-edge blade, and transferred onto the surface of potato dextrose agar (PDA; 200 g potatoes, 20 g dextrose, 20 g agar per L) and malt extract agar (MEA; 30 g malt extract, 5 g mycological peptone, 15 g agar per L), and incubated at 25 ◦C to obtain the pure culture. After 7 days of incubation, greyish fungal colonies appeared on PDA. Single-spore isolation method was employed to recover the pure cultures for six isolates. The colonies initially produce light gray aerial hyphae, which turn dark gray as they mature. Conidiophores (n=50) single or in small groups, straight or curved, sometime geniculate, 20-50 nm long, with scars. Conidia (n=50) were obclavate to pyriform and measured 15 to 60 μm long, 4 to 16 μm wide with 0 to 3 longitudinal, and 1 to 6 transverse septa with short beak (2-30 μm). The morphological characters matched those of Alternaria alternata (Simmons 2007). DNA was extracted from the fungal colonies using a Ezup Column Fungi Genomic DNA Purification Kit. The internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation elongation factor 1-alpha (tef1) were amplified with the primers ITS1/ITS4 (White et al. 1990), gpd1/gpd2 (Berbee et al. 1999) and EF1-728F/EF1-986R (Carbone & Kohn 1999). The obtained sequences were deposited in the GenBank (ITS: OM759881 and OM759882, GAPDH: ON376732 and ON376733, tef1: ON376730 and ON376731). The morphological characteristics and molecular analyses of the isolate matched the descriptin of A. alternata. To perform pathogenicity test, The seedlings of twenty 2-year-old potted H. mutabilis plants were inoculated by spraying conidial suspension at the concentration of 1 × 106 conidia/ml on both sides of leaves and ten plants sprayed with sterile water served as control. The test was repeated three times. All plants were covered with polyethylene covers and kept under the greenhouse at 26 ± 1 ℃. After six days, the inoculated plants showed the same symptoms as the original diseased plants and the controls remained asymptomatic. The fungal pathogen was reisolated from the artificially infected plants and confirmed as A. alternata based on morphocultural characteristics and PCR assays. The results indicated that A. alternata is a causal agent of the disease. The leaf spot disease of cotton rose caused by Nigrospora oryzae has already been reported from Sichuan, China (Han et al. 2021). To our knowledge, this is the first report on the presence of A. alternata affecting H. mutabilis plants. The identification could provide relevant information for adopting appropriate management strategies to control the disease.

Identification of Neofusicoccum parvum as the causative agent of leaf spot disease on Bletilla striata in China.

Bletilla striata (Thunb.) Rchb. f. (Orchidaceae) is an essential traditional Chinese medicinal plant used to treat hemorrhage, swelling, inflammation, ulcers, and pulmonary diseases (Xu et al. 2019). In April of 2020, an unknown leaf spot disease was observed on B. striata in a plantation (~ 0.2 ha) in Nanning, Guangxi province, China. Disease incidence was estimated at approximately 25% (n = 150 plants). The initial symptoms were small brown circular spots, which then expanded into reddish to brown, circular to irregular lesions 5-10 mm in diameter. As the disease developed, the whole leaf became densely covered with lesions. Finally, the lesions coalesced, killing the leaf and resulting in defoliation. To isolate the causal agent, six symptomatic leaves were collected from individual plants. Small pieces (~ 5 mm2) were cut from the margin of the necrotic lesions (n = 18), disinfected in 1% NaOCl for 2 min before rinsing three times in sterile water, and placed on potato dextrose agar (PDA) at 26°C for 3 days. Hyphal tips from the resulting cultures were transferred to PDA to obtain pure cultures. Fifteen isolates were obtained, of which twelve isolates exhibited similar morphology. Colonies on PDA were initially white, then turned dark gray after 7 days. Pycnidia were produced on the surface of PDA after 50 days. Conidia were hyaline, aseptate, ellipsoidal to fusiform, externally smooth, thin-walled, and measuring 11.5 to 15.2 × 4.9 to 6.1 μm (mean ± SD: 13.4 ± 1.0 × 5.4 ± 0.3 μm, n = 60). Morphological features were similar to N. parvum (Phillips et al. 2013). For further molecular identification, the internal transcribed spacer (ITS) region, partial translational elongation factor subunit 1-α (EF-1α), β-tubulin (TUB2) genes were amplified and sequenced using the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F (Carbone and Kohn 1999)/EF-2 (O'Donnell et al. 1998), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. Sequences of the two isolates BJ-111.1 and BJ-111.4 were deposited in NCBI GenBank under the following accession numbers: OM348509-10, OM397537-40. The obtained ITS, EF1-α, and TUB2 sequences showed 99% (514/516, and 513/516 bp), 99% (275/276, and 274/275 bp), and 99% (429/431, and 429/430 bp) homology with several GenBank sequences of the ex-type strain N. parvum CMW 9081 (AY236943, AY236888, and AY236917, respectively) (Zhang et al. 2017). In addition, a phylogenetic analysis confirmed the isolates as N. parvum. Therefore, the isolates were identified as N. parvum based on morphological and molecular evidence. Furthermore, pathogenicity tests were carried out on 1.5-year-old B. striata plants. Healthy leaves on six plants (1 leaf per plant) were inoculated with a 10-μl droplet of conidial suspensions (106 conidia/mL). Three plants treated with sterile water served as the control. All plants were covered with transparent plastic bags and incubated in a greenhouse at 26°C with a 12 h photoperiod. Six days post-inoculation, the inoculated leaves showed leaf spot symptoms, while the control plants remained healthy. The experiments repeated three times showed similar results. Finally, N. parvum was consistently re-isolated from the infected leaves and confirmed by morphology and sequencing, fulfilling Koch's postulates. No fungus was isolated from the controls. To our knowledge, this is the first report of N. parvum causing leaf spot of B. striata worldwide. This result will help develop disease management strategies against this pathogen.

Isolation and potential biocontrol of the fungus causing anthracnose in longan in Vietnam

Introduction: Longan is a crop plant of very high economic value, and both its fruit and flower are beneficial for human health. Longan has been increasingly cultivated in Vietnam due to the increasing demand for domestic consumption as well as export of its fruit. However, the widespread emergence and spread of anthracnose, a group of fungal disease affecting a wide range of plant species, in longan has seriously affected both the longan fruit yield and quality in Vietnam. Current methods for the prevention of anthracnose in longan depend mainly on the use of fungicides which are very harmful to human health as well as disruptive to microbial community structures in different ecosystems. In order to obtain an environmentally friendly method of control for this disease, the agent causing anthracnose in longan must first be identified. Therefore, the aim of this study was to isolate and identify the causal anthracnose agent in longan in Vietnam.
 Methods: Experiments were first carried out with pieces of anthracnose longan leaves pressed onto the surface of potato-dextrose agar (PDA) and incubated for days at 30◦C. Colonies, varying in appearance, were repeatedly isolated and purified on PDA agar, and the anthracnose-causing agent was initially recognized on the basis of colony characteristics and cell morphology. The suspected isolate was then tested for its ability to decompose healthy longan leaf in vitro, and its rDNA region was cloned and sequenced to determine its taxonomy. Antifungal activity testing was performed using the co-culture method.
 Results: We obtained a fungal isolate with septate hyphae, ovoid appressoria, and conidia (which were cylindrical in shape with rounded ends). This isolate showed a clear ability to decompose healthy longan leaves. At the molecular level, the isolate was determined to be a fungal species belonging to genus Colletotrichum, and therefore named Colletotrichum sp. strain BKHCM. We also found that its growth was inhibited when co-cultured with Streptomyces flaveus, an actinomycete originating from soil.
 Conclusion: For the first time, we isolated a fungal species belonging to genus Colletotrichum from anthracnose-infected longan leaves in Vietnam. We also showed that the growth of this fungus could potentially be biocontrolled.

First Report of Neofusicoccum parvum Causing Fruit Rot on Eriobotrya japonica in China

Loquat, Eriobotrya japonica (Thunb.) Lindl., is one of the most economically important fruit trees in China. During 2017, a field survey conducted in orchards of the Fuling District, Chongqing City, revealed the occurrence of fruit rot of mature fruit of E. japonica ‘Dawuxing’. Water-soaked decay with a brown stripe around the lesion was present on symptomatic fruits. Tissues from symptomatic fruit (5 × 5 mm) were aseptically separated and surface disinfested in 2.5% sodium hypochlorite for 45 s, rinsed in sterile water, disinfested in 75% ethyl alcohol for 45 s, rinsed in sterile water twice for 30 s, placed onto potato dextrose agar (PDA), and incubated at 25°C in the dark for 3 days. Tissues from 30 out of 32 affected fruits developed fungal colonies. Seven of them showed a white mycelium, moderately dense, becoming dark gray after 5 days. Pycnidia of strain YLLO2-1a were produced on the surface of PDA after 50 days under a photoperiod of 12 h at 25°C. Conidia were hyaline, aseptate, ellipsoidal, measuring (12.4 to) 15.8 (to 19.4) × (4.5 to) 6.7 (to 9.4) μm. Based on morphological characteristics, strain YLLO2-1a was tentatively identified as Neofusicoccum sp. (Crous et al. 2006; Palavouzis et al. 2015; Slippers et al. 2004). Further species identification was performed by polymerase chain reaction and BLAST sequencing analyses of the partial internal transcribed spacer (ITS) rDNA region, β-tubulin (BT), and translation elongation factor 1 alpha (EF-1α) genes. Sequences of strain YLLO2-1a have been deposited in GenBank under accession numbers MH393618 (ITS region), MH393617 (BT gene), and MH824423 (EF-1α gene). BLAST searches of those sequences in GenBank showed >99% similarity with sequences of reference N. parvum isolate CMW9080 (ITS, accession no. AY236942; BT, accession no. AY236916; and EF-1α, accession no. AY236887). To confirm pathogenicity, mycelium plugs of a 3-day-old colony of strain YLLO2-1a were placed onto the surface of wounded and unwounded mature fruits (six replicates) of loquat ‘Dawuxing’. Wounded and unwounded but not inoculated fruits were used as controls. The experiment was repeated twice. Inoculated fruits were incubated at 25°C in plastic containers (42.0 × 31.0 × 12.0 cm) covered with plastic film. All inoculated fruits showed small necrosis spots at 24 h postinoculation (hpi). The rot lesions quickly expended into the entire fruit at approximately 96 hpi. Both inoculated wounded and unwounded fruits developed lesions of 5.75 ± 0.88 and 4.33 ± 0.25 cm² (average area ± SD) at 72 hpi, respectively, whereas the control fruits inoculated with noncolonized PDA plugs remained asymptomatic. The pathogen was reisolated from the lesions of inoculated fruits and confirmed to be N. parvum by morphological characterization and ITS sequence. Based on these results, N. parvum was determined as the pathogen responsible for the fruit rot disease on loquat. N. parvum is a broadly spread pathogen, causing cankers and dieback on loquat tree in Italy and Spain (Giambra et al. 2016; Gonzalez-Dominguez et al. 2017). However, shoots and branches infected with N. parvum were not found in our study. To our knowledge, this is the first report of N. parvum affecting loquat in China.

Inactivation of Penicillium digitatum and Penicillium italicum under in vitro and in vivo conditions by using UV-C light.

In this study, the effects of UV-C on two of the main wound pathogens of citrus fruits, Penicillium digitatum and Penicillium italicum, were investigated with different inoculation methods in vitro and on oranges. P. digitatum and P. italicum spores were inoculated onto the surface of potato dextrose agar or oranges using spread, spot, wound, and piercing inoculation methods. UV-C treatment for 1 min from a working distance of 8 cm reduced the numbers of P. italicum and P. digitatum by about 3.9 and 5.3 log units, respectively, following spread inoculation under in vitro conditions. Significant reductions were obtained after 1-min UV-C treatments of the tested fungi following inoculation using the spread and spot methods. With inoculation by the wound and piercing methods, the tested spores were not inactivated completely even after 10- and 20-min treatment times, respectively. The application of UV-C (7.92 kJ m(-2)) on oranges reduced the percentage of oranges infected at least threefold compared with the rate of infection in the untreated control samples. UV-C irradiation could effectively inactivate spores of P. italicum and P. digitatum inoculated by the spread plate and spot inoculation methods under in vitro and in vivo conditions. On the other hand, because of the low penetration ability of UV-C light, the tested fungi were not completely inactivated following inoculation with the wound and piercing methods. UV-C treatment has potential for use in surface decontamination of citrus fruits.

First Report of Fruit Rot on Schisandra chinensis Caused by Penicillium glabrum and P. adametzii in China.

Schisandra (Schisandra chinensis (Turcz.) Baill) is an important medicinal herb in China, which is mainly used for treatment of insomnia and memory decay. In September 2010, rot was observed on approximately 5% of the fruits during ripening of schisandra in several orchards at Linjiang City and Ji'an City, Jilin Province. Watery spots on infected fruits of schisandra appeared at the end of ripening, and then the fruits darkened in color, shrunk, and turned soft. The surface of the lesions became covered with masses of blue-green mycelium, conidiophores, and conidia under high humidity. To isolate the causal agent, conidia and conidiophores were suspended in sterile water and streaked onto the surface of potato dextrose agar (PDA). Single hyphal tips were then transferred to new PDA plates to be purified. The isolates were then cultured on CYA (Czapek yeast extract agar) and two kinds of strains (wwzqm1 and wwzqm2) were established based on differential growth rate, microscopic features, and colony color. Pathogenicity of each strain was tested on 25 healthy mature fruits of schisandra cv. Red Pearl by inoculating the fruit surface with a 15 μl conidial suspension (106 conidia/ml). Control fruits were treated with sterile water. Fruits were kept at 25°C and 90% relative humidity. After a 5-day incubation, symptoms described above were observed on all inoculated fruits, whereas all control fruits were symptomless. The causal agent was reisolated, confirming Koch's postulates. Strain wwzqm1 was identified as Penicillium glabrum (Wehmer) Westling on the basis of its morphology. Conidiophores arose from basal hyphae, with stipes smooth or finely roughened, 50.5 to 300.0 × 2.5 to 3.5 μm, penicillus monoverticillate, and bearing verticils of 8 to 12 phialides. Conidia were globose to subglobose, approximately 2.9 to 3.5 μm in diameter, with smooth or nearly smooth walls, and conidial chains in compact columns. Colonies grown for 7 days on CYA at 25°C attained a diameter of 32.4 to 39.1 mm, with the center of a deep bluish green, plane, velutinous, and heavily sporulated. Margin mycelium of the colony was white, with a yellowish brown under surface. Strain wwzqm2 was identified as P. adametzii K. M. Zalessky according to its morphological features with conidiophores born from funicolose hyphae, stipes smooth, 10.2 to 20.1 × 1.5 to 2.5 μm, penicillus monoverticillate, and bearing verticils of 4 to 6 phialides. Conidia were nearly globose to glubose, 2.0 to 2.7 μm in diameter, with smooth or finely roughened walls, and conidial chains loose and irregular. Colonies on CYA at 25°C for 7 days grew rather fast and reached a diameter of 50.3 to 60.2 mm, and were deep grayish green, near plane, floccose or funicolose, and heavily sporulated. Margin mycelium of the colony was white with a yellowish brown under surface (1). The internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) was amplified for wwzqm1 and wwzqm2 using primers ITS4/ITS5 and sequenced. BLASTn analysis of the 595 bp wwzqm1 (GenBank Accession No. JN887323) amplicon had 99% sequence identity with P. glabrum (DQ681321) and the 568 bp wwzqm2 amplicon (JN887322) had 99% sequence identity with P. adametzii (AF033401). To our knowledge, this is the first report of fruit rot on S. chinensis caused by P. glabrum and P. adametzii in China. Reference: (1). H. Z. Kong. Penicillium et Teleomorphi Cognati. Flora Fungorum Sinicorum. Science Press, Beijing, 35:43, 2007.

Microencapsulation of hexanal by radiant energy vacuum microwave-molecular inclusion for controlled release and inhibition of Penicillium expansum in a model system and on apple tissue

The shelf-life of apple slices is limited due to rapid decay caused by the growth of fungi such as Penicillium expansum. The purpose of this work was to examine the controlled release of hexanal for the inhibition of P. expansum in a model system and on fresh-cut apple. Hexanal was microencapsulated in β-cyclodextrin–pectin blends by radiant energy vacuum (REV) drying and conventional freeze drying. Inhibition of P. expansum was examined by measurement of radial growth on the surface of potato dextrose agar in petri dishes incubated for 5days at 25°C and 10days at 12°C. Apple slices inoculated with P. expansum spores were stored for 15days at 5°C and 12days at 12°C in sealed glass jars. Hexanal release profiles were quantified using solid phase microextraction gas chromatography (SPME-GC). Release of hexanal from the microcapsules delayed P. expansum spore germination and radial growth on PDA at both temperatures. The visual quality of apple slices stored with microencapsulated complexes was improved when compared to a negative control for up to 15days at 5°C, showing a visual quality rating of 5.67–6.00, while the negative control had a rating of 2.00. The growth of P. expansum applied to the flesh was inhibited by the inclusion of microcapsules dried by lyophilization or REV drying. Encapsulation of hexanal in β-cyclodextrin–pectin blends may provide a means to extend the shelf-life of this highly perishable product.

First Report of Botrytis Leaf Blight and Fruit Rot on Schisandra chinensis Caused by Botrytis cinerea in China.

Schisandra (Schisandra chinensis (Turcz.) Baill) is a perennial plant belonging to Magnoliaceae. It is a very important medicinal herb in China and is mainly used for treatment of insomnia and memory decay. From July to September 2008, an unknown leaf blight and fruit rot on schisandra were first observed at Jingyu County, Jilin Province. The same symptoms were detected in other areas of Jilin Province, such as Ji'an City, Baishan City, and Hunchun City. Initially, some small, brown spots appeared on the tip or margin of the leaves. Light brown or brown necrotic lesions developed and eventually covered entire leaves. Seriously affected leaves were rolled or distorted and eventually became completely dry and brittle. Small spots appeared on the surface of mature fruits, coalesced, and the fruits finally dropped. Gray mycelia and conidiophores developed on the diseased leaves and fruits. To isolate the causal agent, conidia and conidiophores were scraped aseptically from the internal tissues, suspended in sterile water, and streaked onto the surface of potato dextrose agar (PDA). Single-hyphal tips were transferred on PDA and the isolated fungus was identified as Botrytis cinerea Pers.: Fr. on the basis of its morphological characteristics and internal transcribed spacer (ITS) sequence. Colonies of B. cinerea on PDA were colorless at first and became gray to brown 20 days later with the mycelium growth and conidia producing in cultures. Conidia are single celled, lemon shaped, colorless to a light color, and 4.4 to 15.0 × 7.0 to 10.0 μm. Sclerotia formed about 1 week later, were black-brown and varied in size (2.0 to 5.0 × 2.0 to 4.0 mm) and shape. The ITS region of rDNA was amplified from DNA extracted from single-spore isolate BC12 of B. cinerea using primers ITS4/ITS5 and sequenced (GenBank Accession No. GU724512), BLAST analysis (1) of the 535-bp segment showed 99% similarity with the sequence of Botryotinia fuckeliana (perfect stage of B. cinerea). Pathogenicity tests were carried out on healthy schisandra plants that were 4 years old. After the surface of the leaves and fruits was disinfected with 5% sodium hypochlorite, a conidial suspension of 105 conidia/ml was sprayed on 10 schisandra leaves, and plugs of the fungus obtained from the colony margins were transferred onto a 3- × 3-mm wound on the surface of disinfected fruit. Ten control schisandra leaves and 10 fruits were inoculated at the same time. Plants were covered with polyethylene bags and incubated at 25°C in a greenhouse with relative humidity of 85% for 3 days. Similar symptoms to those observed on diseased leaves and fruits in the field were observed on inoculated schisandra leaves and fruits 7 days after inoculation, whereas control leaves and fruits showed no symptoms. The pathogen was successfully reisolated. The gray mold disease caused by B. cinerea was reported in many plants, such as Lavandula stoechas and Chamelaucium uncinatum in Italy (2,3). However, to our knowledge, this is the first report of gray mold disease of schisandra caused by B. cinerea in China.

First Report of Botrytis cinerea Causing Gray Mold of Pomegranate (Punica granatum) in Greece.

Pomegranate is rapidly increasing in production in Greece. During August of 2008 in the region of Larisa (central Greece), preharvest fruit rot was observed on pomegranate (cv. Kapmaditika) that caused losses estimated at 10%. Symptoms first appeared as small spots on the fruits that later increased in size and developed into expanded, dark brown lesions. Internally, tissues were soft and brown with gray mycelia and conidiophores observed. Affected fruits decayed completely during 2 months of storage (5 to 6°C), causing yield losses of up to 20%. To isolate the casual agent, conidia and conidiophores were scraped aseptically from the internal tissues, suspended in sterile water, and streaked onto the surface of potato dextrose agar (PDA). Single hyphal tips were transferred to PDA, and the isolated fungus was identified as Botrytis cinerea Pers.:Fr. on the basis of morphological characteristics (2). B. cinerea was consistently isolated from symptomatic tissues. Colonies of B. cinerea on PDA were at first colorless and became gray to brown with the development of lemon-shaped conidia (average 7.5 × 9 μm). Sclerotia were black and varied in size (1.4 to 4.5 × 1.5 to 2.7 mm) and shape (2). Pathogenicity of the isolated fungus was tested by wound inoculating five mature pomegranate fruits (cv. Kampaditika) after surface sterilization with 5% sodium hypochlorite. Plugs of the fungus (5 mm in diameter) obtained from the colony margins were transferred onto a 3- × 3-mm wound on the surface of sterilized fruit. Sterile PDA plugs were used to inoculate five control pomegranate fruits. Fruit were incubated at 22°C and 80% relative humidity in the dark. Extensive decay, similar to that observed on diseased fruits in the field, was observed on inoculated fruits 7 days after inoculation, whereas control fruits showed no decay. The pathogen was reisolated from internal rotten tissues of inoculated fruit, but not from the noninoculated control fruits. Fruit rot of pomegranate caused by B. cinerea has been reported previously in the United States (1) and China (3). However, to our knowledge, this is the first report of B. cinerea causing gray mold of pomegranate in Greece.

First Report of Canker Disease Caused by Botryosphaeria parva on Cork Oak Trees in Italy

A survey was carried out in the spring of 2003 to study the fungi associated with declining trees in a cork oak (Quercus suber L.) forest located in Sassari Province, Sardinia, Italy (40°52'N, 9°01'E) at an altitude of 150 m (above sea level). Several isolates obtained from live twigs and branches showing sunken necrotic bark lesions were identified as Fusicoccum parvum Pennycook & Samuels (teleomorph Botryosphaeria parva Pennycook & Samuels). Neither pycnidia nor ascomata were observed on the symptomatic samples collected. On potato dextrose agar (PDA) at 25°C, the isolates developed an aerial and compact mycelium, initially white but becoming gray after 4 to 6 days, and produced pycnidia after 1 month on sterile cork oak twigs placed on the surface of PDA. Conidia from culture were hyaline, ellipsoidal to fusiform, externally smooth, thin walled, nonseptate, 12 to 19 (15.5) × 5.5 to 8.5 (6.5) μm, with length/width ratio of 2.4 ± 0.1 (mean ± standard error). Identity was confirmed by analysis of the nucleotide sequences of the internal transcribed spacer (ITS) from the rRNA repeat and the translation elongation factor 1-alpha (EF1-α), as done elsewhere (1,4). BLAST searches at GenBank showed a high identity with reference sequences (ITS: >99%; EF1-α: 100%). Representative sequences of both regions were deposited at GenBank (ITS: Accession No. DQ487157; EF1-α: Accession No. DQ487158). Pathogenicity tests were carried out on seven 2-year-old cork oak seedlings maintained in a greenhouse at 14 to 26°C with the B. parva strain CBS 119937 obtained in this study. A mycelial plug (3 to 4 mm2) taken from the margin of an actively growing colony on PDA was put in a shallow wound made by a scalpel on the basal part of the stem of each seedling. Sterile PDA plugs were placed into similar wounds on three control seedlings. The inoculation points were wrapped in Parafilm to retain moisture for 1 week. After 4 weeks, all seedlings inoculated with B. parva died and showed a collapse of the stem cortical tissues associated with dark brown discolorations and vascular necrosis measuring 10.9 ± 0.4 cm. No symptoms were visible in the control seedlings. The pathogen was reisolated from all the inoculated seedlings, thus fulfilling Koch's postulates. The results confirm the virulence of this fungus and point to its possible involvement in the aetiology of cork oak decline. B. parva is a cosmopolitan, plurivorous pathogen causing disease in several hosts of economic importance, such as grapevine (3), kiwi (2), and Eucalyptus spp. trees (1). To our knowledge, this is the first report of B. parva causing canker disease on cork oak trees.

First Report of Phomopsis amygdali Causing Fruit Rot on Peaches in Greece.

In the summer of 2005, the fungus Phomopsis amygdali (Del.) Tuset & Portilla was frequently isolated from decayed peaches (Prunus persica cv. Andross) grown in the province of Imathia, Greece. Fruit infected by P. amygdali developed gray-to-brown decay lesions with white mycelium forming on the surface of lesions. Identification of the pathogen was based on morphological characteristics. Dark-pigmented pycnidia (flask-shaped, conidia-bearing fruiting bodies) were produced over the surface of potato dextrose agar. The pycnidia exuded conidia in white tendrils 7 days later. Koch's postulates were completed in the laboratory by inoculating mature and immature cv. Andross peach fruits with an isolate of P. amygdali isolated from decayed cv. Andross peaches. Thirty peach fruit were surface sterilized by dipping them into 0.1% chlorine solution and allowing them to dry in a laminar flow hood. The peach fruit were wounded with a 2-mm diameter glass rod and a 40-μl drop of 5 × 105 conidia of P. amygdali per milliliter suspension was applied to the wound. Thirty control fruits were similarly wounded and inoculated with a 40-μl drop of sterile water. All inoculated and noninoculated fruit were incubated at 24 to 26°C for 7 days. Koch's postulates were satisfied when the same fungus was reisolated from 100% of inoculated mature and immature fruit that developed symptoms similar to diseased fruit collected from orchards. Although P. amygdali has been previously reported as a causal agent of canker disease (2) and fruit rots of peaches (1) in other countries, to our knowledge, this is the first report of the occurrence of P. amygdali causing a fruit rot of peaches in Greece.

Tissue Necrosis of Sour Cherry Fruits, An Unusual Disease Occurrence Caused by a Phoma sp. in Serbia.

Following frequent rains during May and June of 1999, a high incidence of an unknown disease on sour cherry (Prunus cerasus) fruits was observed in western Serbia (Šabac). Initially, small, water-soaked, dark green spots appeared on the fruit tissue similar to infections caused by Pseudomonas syringae or a Colletotrichum sp. (1,2). Later, spots enlarged, coalesced, and became necrotic and covered most of the fruit surface. The diseased tissue was slightly depressed and fruit shriveled and dried. Approximately 60 to 80% of the total yield was affected. Numerous hyaline, unicellular, and ellipsoidal conidia were observed with a microscope. Orange spore masses often occurred on the necrotic tissue following rain. Symptoms of the disease were not expressed during the dry spring of 2000, but were observed in rainy 2001. The most susceptible cultivar was Haiman's Rubin. Disease symptoms were absent on the domestic cultivar Oblačinska. Placing small necrotic fragments on potato dextrose agar (PDA) resulted in grayish fungal colonies that produced black, globose pycnidia with hyaline, unicellular, and ellipsoidal conidia under daylight conditions that were similar to those formed on necrotic sour cherry fruit in the orchard. At 25°C, colonies covered the entire 9-cm diameter surface of PDA after 5 to 6 days. No teleomorph was observed. Bacterial isolations on nutrient agar were unsuccessful. Unripe sour cherry fruits were punctured with a sterile needle and small colony fragments of the fungus were placed over the wound. Symptoms typical of those on fruit found in orchards were observed after 7 days of incubation at 25°C. No symptoms developed on control fruits punctured with a sterile needle. The fungus was reisolated from the inoculated fruits and shown to be identical to the original strains. On the basis of pathogenicity and morphological characteristics of the strains investigated, the fungus was determined to be a Phoma sp. (3), also known as sour cherry necrosis in other fruit-tree-growing regions. To our knowledge, this is the first report of the fungus on sour cherry fruit in Serbia.

Induction of Chlamydospores in Trichoderma harzianum and Gliocladium roseum by Antifungal Compounds Produced by Bacillus subtilis C2

AbstractOne bacterial strain was isolated from a Panax notoginseng field and identified as Bacillus subtilis C2 by its physiological and biochemical characteristics. The culture filtrate of the bacterium showed strong antifungal activity against Trichoderma harzianum and Gliocladium roseum. The antifungal compound termed 10M was purified from the culture filtrates of B. subtilis C2 through organic solvent extraction, silica gel H and Sephadex LH‐20 column chromatography. Chlamydospores were formed either inside the conidia or from hyphae, when the conidia of T. harzianum and G. roseum were cultured on the surface of the potato dextrose agar (PDA) containing relatively low concentrations of 10M for 48 h. While they were cultured on the surface of PDA containing relatively high concentrations of 10M for 48 h the cells were swollen and then lysed. The 10M had high similarity to iturin A as showed by matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. This is the first report that antifungal compounds produced by B. subtilis can induce chlamydospore formation in biocontrol fungi at relatively low concentrations.

A Method for Purifying Cochliobolus sativus Cultures Contaminated with Bacteria

A simple method was developed for purifying Cochliobolus sativus cultures contaminated with bacteria. In this method the contaminated fungal culture is scraped from the surface of the potato dextrose agar (PDA) growth medium. Small leaf pieces (approximately 0.5 cm) of a highly susceptible genotype of barley were placed on the scraped surface of PDA and incubated for 72 h. Disease symptoms of C. sativus were detected on the leaf pieces. Colonies that developed from these pieces on new PDA medium were free from bacteria.

First Report of Rhizoctonia solani AG-4 and AG-2-2 on Lupinus angustifolius in Canada.

Narrow-leaved lupine (Lupinus angustifolius L.) is grown as a grain legume crop in Australia and Europe where it is used as feedstock in the livestock and aquaculture industries. During July 2003, a stem rot disease was observed in narrow-leaved lupine (cv. Arabella) plants in a research plot at the Crop Diversification Centre North (CDCN), Edmonton, Alberta, Canada. The disease was also found on cv. Rose at the CDCN and cv. Arabella in experimental fields near Devon, Ellerslie, and Westlock, Alberta during the late spring of 2004. Diseased plants showed dark brown-to-black stems with sunken and constricted lesions at the soil level. Young leaves became shrunken and twisted and seedlings collapsed. Rhizoctonia solani was consistently isolated from lesions on taproots and basal stems of diseased plants. Colonies of cream-colored mycelia grew close to the surface of potato dextrose agar (PDA). Most sclerotia formed inside the medium. Agar disks (1 cm in diameter) of isolates LP-11Bb, LP-24C, and LP-25C were attached to the opposite sides of basal stems (180° apart) of 1-month-old lupine seedlings (cv. Arabella). Inoculated plants were incubated for 2 days in black plastic bags under a greenhouse bench at approximately 20°C. All isolates caused brown lesions on the lower stems (extending up to 7 cm above ground level), girdling, and root rot. Plants wilted within 7 to 10 days after inoculation, and aerial mycelia appeared on the basal stems. R. solani was reisolated from the infected crown tissues using PDA to complete Koch's postulates. The isolates were paired with AG tester strains of R. solani. Isolates LP-11Bb and LP-24C were identified as AG-4 while isolate LP-25C was identified as AG-2-2. In another trial, eight isolates of R. solani (unknown AG types) were tested for virulence on L. angustifolius cv. Arabella using the inoculation method described above. All isolates were pathogenic, and disease severity that was based on a 0 to 4 scale ranged from 2.7 to 3.2. The most virulent strain was LP-24C, which caused a 77% loss in fresh weight compared with the noninoculated control plants. R. solani AG-8 is associated with Rhizoctonia disease of lupine in Australia (1) and also causes bare patch disease of wheat. To our knowledge, this is the first report of R. solani on lupine in Canada. This disease could have a significant impact on the commercial production of lupine in Alberta. Reference: (1) M. W. Sweetingham et al. Pages 466-486 in: Advances in Lupin Disease Management in Australia. Proc. Int. Lupin Conf., 8th. G. D. Hill, ed. International Lupin Association, Canterbury, New Zealand, 1999.

Characterization of a New Subgroup of Rhizoctonia solani Anastomosis Group 1 (AG-1-ID), Causal Agent of a Necrotic Leaf Spot on Coffee

ABSTRACT A new foliar disease on coffee leaves was observed in Mindanao, Philippines, in 1996. The symptoms appeared as large circular or irregularly shaped necrotic areas with small circular necrotic spots (1 mm or less in diameter) usually found around the periphery of the large necrotic areas. Rhizoctonia solani was consistently isolated from these diseased coffee leaves. Isolates obtained were multinucleate (3 to 12 nuclei per hyphal cell), had an optimum temperature for hyphal growth at 25 degrees C, prototrophic for thiamine, and anastomosed with tester isolates belonging to R. solani anastomosis group 1 (AG-1). Mature cultures on potato dextrose agar (PDA) were light to dark brown. Sclerotia, light brown to brown, were formed on the surface of PDA and covered the whole mature colony culture. Individual sclerotia often aggregated into large clumps (3 to 8 mm in diameter) and their color was brown to dark brown. In pathogenicity tests, isolates from coffee caused necrotic symptoms on coffee leaves, whereas isolates of AG-1-IA (not isolated from coffee), 1-IB, and 1-IC did not. The results of analyses of restriction fragment length polymorphism of ribosomal DNA internal transcribed spacer, random amplified polymorphism DNA, and fatty acid profiles showed that R. solani isolates from coffee are a population of AG-1 different from AG-1-IA, 1-IB, and 1-IC. These results suggest that R. solani isolates from coffee represent a new subgroup distinct from AG-1-IA, 1-IB, and 1-IC. A new subgroup ID (AG-1-ID) is proposed.

Fusarium moniliforme (Sheldon) isolated from gills of kuruma prawn Penaeus japonicus (Bate) with black gill disease.

Fusarium moniliforme was isolated from gill lesions of kuruma prawn Penaeus japonicus, with black gill disease at a private farm in Okinawa Prefecture in 1989. The colonies of the fungus cultured on upper surface of potato dextrose agar were floccose, creamy white, undersurface a lavender to violet, but did not grow on mycobiotic agar containing cycloheximide. The present report describes the first case of F. moniliforme infection in crustacea. An experimental infection using kuruma prawn was made by intramuscular injection with the conidia of F. moniliforme NJM 8995. For comparison, Fusarium solani NJM 8996 isolated also from a kuruma prawn with black gill disease in Okinawa Prefecture in 1989, was used as a reference. The clinical signs and path-ological findings of the disease caused by the two species of the fungi were similar. Identification of the fungi isolated from the lesions was based principally upon the cultural characteristics. The use of a media lacking cycloheximide is recommended for the isolation of F. moniliforme.

A loculate stromatic conidiomatal isolate of Glomerella cingulata

An isolate of Glomerella cingulata (anamorph Colletotrichum gloeosporioides) which formed loculate stromatic conidiomata was recovered from a single ascospore line from soybeans (Glycine max). The loculate stromatic conidomata were formed on the surface of potatodextrose agar and subepidermally in apple fruits. The loculi were without ostioles and broke open presumably from pressure exerted by developing conidia. Conidiogenesis and conidial morphology are similar to ascospore lines of three other acervular anamorphs of G. cingulata.