As a polyene antibiotic of great pharmaceutical significance, pimaricin has been extensively studied to enhance its productivity and effectiveness. In our previous studies, pre-reaction state (PRS) has been validated as one of the significant conformational categories before macrocyclization, and is critical to mutual recognition and catalytic preparation in thioesterase (TE)-catalyzed systems. In our study, molecular dynamics (MD) simulations were conducted on pimaricin TE-polyketide complex and PRS, as well as pre-organization state (POS), a molecular conformation possessing a pivotal intra-molecular hydrogen bond, were detected. Conformational transition between POS and PRS was observed in one of the simulations, and POS was calculated to be energetically more stable than PRS by 4.58 kcal/mol. The structural characteristics of PRS and POS-based hydrogen-bonding, and hydrophobic interactions were uncovered, and additional simulations were carried out to rationalize the functions of several key residues (Q29, M210, and R186). Binding energies, obtained from MM/PBSA calculations, were further decomposed to residues, in order to reveal their roles in product release. Our study advanced a comprehensive understanding of pimaricin TE-catalyzed macrocyclization from the perspectives of conformational change, protein-polyketide recognition, and product release, and provided potential residues for rational modification of pimaricin TE.

An animal model of keratomycosis was used to study the interaction between the new enzyme antifungal compound mycolase II and the polyene antifungal antibiotic pimaricin. Well established corneal infection caused by an ocular pathogenic candida albicans on New Zealand white male rabbits were treated with 3% mycolase II, 5% pimaricin and a combination of 3% mycolase II and 5% pimaricin respectively. The rates of resolution of the corneal lesions for each group of eyes treated by the various drugs were determined and the results were analysed by computer using a two-way analysis of variance to determine the interaction or independence of 3% mycolase II in combination therapy with 5% pimaricin in rabbit keratomycosis. The analysis of variance showed a significant level of positive interaction after each period of treatment. (P less than 0.001).

The authors described morphological and biochemical properties of twenty strains of Torulopsis glabrata and two strains of T. candida and T. sphaerica, mostly of human origin. By means of eight biotyping tests based on the evaluation of so-called resistograms the isolates of T. glabrata were divided into eight biotypes. The authors discussed the problem of possible use of biotyping of T. glabrata in investigations of the epidemiology and pathogenesis of mycoses caused by this microorganism. In investigations of the sensitivity of strains of Torulopsis spp. to nine antimycotic agents in vitro it was revealed that the isolates were sensitive to all polyene antibiotics (amphotericin B, nystatin and pimaricin) and to 5-fluorocytosine. The sensitivity to azole chemotherapeutic agents (clotrimazole, econazole, miconazole, ketoconazole and itraconazole) was more varied: some strains were resistant to clotrimazole and econazole.

Two antifungal agents, pimaricin and mycostatin, added to Cottage cheese through the wash water at concentrations of 20, 50, or 100 mug/ml of wash water or added through the cheese dressing at 1, 2, or 5 mug/g retarded the growth of Aspergillus niger and Saccharomyces cerevisiae and improved the shelf-life of the cheese. In general, cheese with highest concentration of antifungal agent and stored at lowest temperature had best keeping quality. Pimaricin was slightly more effective than mycostatin in inhibiting fungi; inhibition was greater if the antifungal agents were added to the cheese dressing and the cheese was stored at low temperature; and A. niger was more sensitive to the inhibitors than S. Cerevisiae.

In fall 2012 and 2013, peanut (Arachis hypogaea L.) grown in commercial fields in Tift County, GA, showed pod rot symptoms. The disease was primarily damaging pods and kernels and symptoms included brown to black, water-soaked lesions on pods and blackened pegs with white fluffy mycelia. Ten random symptomatic pods collected from the field were plated on potato dextrose agar after surface sterilization with 0.5% NaOCl. The plates were incubated at 25°C for 5 days in the dark. Whitish fungus-like cultures with non-septate mycelium grew from all the pods. Single hyphal tip cultures were obtained on pimaricin-ampicillin-rifampicin-pentachloronitrobenzene (PARP) medium. Isolates on PARP agar plates had three different growth patterns: two groups of isolates produced sporangia and the third group produced oogonia. The isolates were identified as Pythium spp. based on growth pattern and sporangial and oogonial structures (2). DNA of one representative isolate from each group was extracted and the internal transcribed spacer (ITS1-5.8S-ITS2) regions of rDNA were amplified and sequenced with primers ITS1 and ITS4 (1). ITS sequences of the isolates shared 99 to 100% similarity with Pythium ultimum, P. vexans, and P. deliense isolates in GenBank (Accession Nos. KC689906, GU133594, and HQ643521, respectively). The isolates were identified as P. ultimum var. ultimum, P. vexans, and P. deliense based on molecular analysis and morphological characteristics. P. ultimum produced plenty of spherical sporangia, but no oogonia in the culture, P. deliense produced characteristic terminal oogonia and aplerotic oospores with oogonial stalks curved towards the antheridia, and P. vexans produced spherical sporangia and aplerotic oospores. ITS sequences of three isolates representing each of the three species were deposited in GenBank (KF500573, KF500574, and KF500572). Pathogenicity of one representative isolate from each group was tested on peanut under greenhouse conditions (30°C day and 20°C night). Nine 10-week-old peanut seedlings (cv. GA07W) grown in 20-cm pots (2:1 ratio of potting mix/sterilized field soil) were inoculated with the isolates separately by applying 5 ml of respective Pythium-infested beet seeds. Nine untreated plants were used as a control. Pods were washed off 1 month after inoculation for disease assessment. All plants inoculated with the isolates showed pod rot similar to those observed in the field. The three Pythium species were re-isolated from respective symptomatic pods and the identity was confirmed by morphological characteristics and molecular analysis. The untreated plants did not show typical pod rot symptoms and the Pythium species were not isolated from these plants. P. ultimum and P. vexans have been reported to be associated with peanut pod rot in the United States (3). To our knowledge, this is the first report of P. deliense causing peanut pod rot. Georgia is the top peanut producer in the United States and the occurrence of pod rot caused by the Pythium spp. needs to be taken into account in developing disease management programs in peanut production. References: (1) M. A. Innis et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990. (2) A. J. Van der Plaats-Niterink. Stud. Mycol. 21:51, 1981. (3) T. A. Wheeler et al. Peanut Sci. 32:9, 2005.

During a 2019-2020 survey for plant pathogenic oomycetes in Nanjing, China, severe foliage blight and dieback were observed on approximately 20 Rhododendron pulchrum plants at three public parks and gardens. Approximately 25% of leaves and shoots were affected. Symptoms included brown to black lesions on leaves and stems, dieback of shoot tips, and wilting. Diseased tissues were collected from a five-year-old shrub with typical disease symptoms at Xuanwuhu Park. They were cut into 10×10 mm2 squares, immersed in 70% ethanol for 30 sec, and placed onto fresh clarified V8 juice agar (cV8A) containing pimaricin, ampicillin, rifampicin, and pentachloronitrobenzene. Phytophthora-like hypae were transferred to new cV8A plates daily. A total of five isolates were obtained after five days of incubation at 25°C. After approximately 20 days, all isolates were identical in morphological traits including semi-papillate sporangia and gametangia (homothallic). Thirty sporangia of a representative isolate Ppi were randomly selected and examined. They were mostly ovoid and sometimes obpyriform, averaging 41.0 ± 3.9 × 24.8 ± 3.2 µm. Antheridia of 30 randomly selected gametangia were paragynous, averaging 16.7 ± 0.7 × 12.4 ± 1.5 µm. Average diameters of oogonia and plerotic oospores were 29.2 ± 0.3 µm and 26.4 ± 1.6 µm, respectively. Chlamydospores were not observed. The above morphological traits suggested the causal agent belonging to the "P. citricola-complex". Isolate Ppi was subjected to sequencing of the rDNA internal transcribed spacer (ITS) region and the ras-related GTP-binding protein 1 (Ypt1) gene. ITS sequence of Ppi (GenBank ACN. MT672594) has 100% identity to that of P. pini (MG865565). It has a 3-nt difference from the ITS sequences of P. acerina (MG518642) and P. citricola (MG865475) and a 4-nt difference from that of P. plurivora (FJ665225). Ypt1 sequence of Ppi (MT680000) has 100% identity to that of P. pini (MK058416). Pathogenicity of Ppi on R. pulchrum was tested using both detached-leaf and whole-plant assays. In the former assay, each of six asymptomatic leaves was symmetrically wounded at both sides using a sterile inoculation needle. A 5×5 mm2 Ppi-colonized cV8A plug was placed on each wound of five leaves. Sterile agar plugs were used for a control leaf. All six leaves were placed on a wet filter paper in a closed container at 25°C. This assay was repeated twice. On the fifth day, all inoculated leaves had necrotic tissues around the wounds, while the control leaves remained asymptomatic. In the whole-plant assay, 20-inch-tall plants were used. Five attached leaves and the twig base of each plant were wounded. A control plant was inoculated in the same manner above, while sterile agar plugs were used. Each plant was covered with a plastic bag and maintained at 25°C. Wet cotton balls were placed in the bags to maintain humidity. After two days, the bag containing cotton balls was removed. This assay was repeated three times. After two weeks, all three inoculated plants in the three replicated trials had severe foliage blight and dieback, whereas control plants remained healthy. Phytophthora isolates recovered from artificially inoculated tissues were identical to isolate Ppi in morphological characters. Rhododendron diseases caused by P. pini were reported in the USA and Finland . This is the first report of P. pini causing foliage blight and dieback on R. pulchrum, an important nursery and landscape plant in China. Additional surveys are ongoing to determine the distribution of this pathogen in Nanjing. Management programs are under development to contain the spread of P. pini and treat diseased plants.

Keywords: Causal Agent; Crop Type; Epidemiology; Oomycetes; Subject Areas; Trees; ornamentals; pathogen survival.

Derivative spectroscopy was used for quantitative determination of natamycin in cheese. When measuring a methanolic cheese extract against methanol, the second derivative of the UV-spectrum is measured between 340 and 290 nm. The natamycin concentration can be determined by measuring the vertical distance between the minimum at 318 nm and the maximum at 311 nm. Under these conditions the detection limit of natamycin in a pure methanolic solution lies at 20 ng/ml, in cheese extracts at 150 ng/ml. The latter corresponds to a natamycin concentration of 2.5 ppm in the case of a 3 g test sample or 0.03 mg/dm2 in the case of a 25 cm2 cheese surface. The introduction of derivative spectroscopy makes it possible to reduce the interference of cheese substances in the photometric measurements and to increase the sensitivity and selectivity of the detection process. Besides the advantage that work and expenses are reduced - as no pimaricin-free sample has to be extracted from the interior of the cheese - it is also possible to determine natamycin photometrically in cheese, in which it is distributed homogeneously.

The paper reports on Candida albicans ocular infection modified by steroid eye drops. A 74-year-old male complained of conjunctival injection and pain in his right eye three months after pterygium and cataract surgery. After treatment with antibiotics and steroid eye drops for three days, he was referred to our hospital. Clear localized corneal endothelial plaque with injection of ciliary body was observed. No erosion of the corneal epithelium, or infiltration of stromal edema was observed, suggesting that the pathological organism derived from the intracameral region. Because ocular infection was suspected, steroid eye drops were stopped, which led immediately to typical infectious keratitis in the pathological region, with epithelial erosion, fluffy abscess, stromal infiltration, and edema. For diagnostic purposes, the plaque was surgically removed with forceps and the anterior chamber was irrigated with antibiotics. The smear and culture examination from the plaque revealed C. albicans surrounded by neutrophils. However, aqueous fluid and fibrous tissue after gonio procedure contained no mycotic organisms. Topical fluconazole, micafungin, and pimaricin with oral itraconazole (150 mg/day) were effective. Special attention is needed when prescribing steroid eye drops to treat corneal disease especially postoperatively. Diagnosing infectious keratitis is sometimes difficult because of modification by some factors, such as postoperative conditions, scarring, and drug-induced masking. Here, we report on mycotic keratitis modified by postoperative steroid administration.

A rare case of fungal keratitis caused by Plectosporium tabacinum is reported. A 78-year-old female gardener presented with conjunctivitis and an oval infiltrate with irregular margins in the nasal half of the cornea in the right eye. Light microscopy of corneal scrapings revealed a filamentous fungus, and a diagnosis of fungal keratitis was made. The patient was admitted into our hospital on February 19, 2008. Treatment with topical miconazole, topical fluconazole, pimaricin ointment, intravenous miconazole, and corneal debridement was commenced. One week later, the infiltrate improved, but the central part of the infiltrate was still deep. Topical fluconazole was switched to topical voriconazole, and intravenous miconazole was switched to intravenous voriconazole. One month after admission, the causative organism was identified by morphology and molecular biological analysis as Plectosporium tabacinum. The corneal infiltrate resolved 3 months after admission. A stromal scar persisted for 3 months after the patient was discharged. This is the first detailed report of fungal keratitis caused by P. tabacinum. Voriconazole was effective in treating this refractory keratitis.

Pythium aphanidermatum, with an optimum temperature for growth at 35C, grew welland was readily isolated from soil on pimaricin-vancomycin medium (MPVM) when incubated for 24h at 38-40C. The pH of the medium affected recovery; maximum numbers developed above pH 6.0. Other Pythium spp. were recovered on MPVM at 20-25C, but were excluded by incubation at 38-40C. These Pythium spp. included P. ultimun, P. paroecandrum, P. irregular, P. mamillatum, and an unidentified Pythium sp. These species grew well and were readily siolated from soil on gallic acid medium (GAM) when incubated for 24-8h at 20 C.P. aphanidermatum and P. myriotylum grew from mycelium on GAM, but their oospores did not germinate nor could they be isolated from soilon this medium. P. myriotylum grew well on MPVM, but was only rarely isolated, evenfrom soils with known high potential for disease caused by P. myriotylum. Propagules of Pythium were enumerated by a plate-dilution frequency method or by a smearplateethod is valuable for studies on the ecology, survival, and inoculum potential in soils with mixed populations of P. aphanidermatum and other Pythium spp.

Petrifilms are dehydrated agar culture plates that have been used to quantify colony forming units (CFU) mL of either aerobic bacteria (Petrifilm-AC) or fungus (Petrifilm-YM), depending on substrate composition. Microbes in irrigation systems can indicate biofilm risk and potential clogging of irrigation emitters. The research objective was to compare counts on Petrifilms versus traditional, hydrated-agar plates using samples collected from recirculated irrigation waters and cultures of isolated known species. The estimated count (in CFU mL) from a recirculated irrigation sample after 7 d of incubation on Petrifilm-YM was only 5.5% of the count quantified using sabouraud dextrose agar (SDA) with chloramphenicol after 14 d. In a separate experiment with a known species, Petrifilm-YM did not successfully culture zoospores of . Isolates of viable zoospores were cultured successfully on potato-dextrose agar (PDA), with comparable counts with a vegetable juice medium supplemented with the antibiotics pimaricin, ampicillin, rifamycin, pentochloronitrobenzene and hymexazol (PARP-H). The quantification of pv. Begoniaceae on Petrifilm-AC was not significantly different ( < 0.05) than on PDA, but was lower than on Reasoner and Goldrich agar (R2A) or with a hemocytometer. The current formulation of Petrifilm-YM is unlikely to be a useful monitoring method for plant pathogens in irrigation water because of the inability to successfully culture oomycetes. However, Petrifilm-AC was an effective method to quantify bacteria and can provide an easy-to-use on-farm tool to monitor biofilm risk and microbial density.

In October 2017, we collected five soil samples from each of several fields with a history of severe corn (Zea mays) seedling disease in Heilongjiang province of China. Affected seedlings were wilted with severe root rot, and a high incidence of seedling death was observed in the fields. Corn seeds were seeded in the collected soil samples and grown in a growth chamber for 21 days set at the following incubation temperatures: 21℃/7℃ for 6 days, 10℃/3℃ for 4 days, 16℃/7℃ for 5 days, 20℃/20℃ for 6 days (16 h/8 h, light/dark) (Tang et al. 2019). The corn seedlings in the growth chamber showed the same symptoms observed in the field as mentioned above. Corn root rot samples were collected from several symptomatic plants in the growth chamber to isolate the possible pathogen. Symptomatic roots were washed in 0.5% NaOCl for 2 min, rinsed in sterile water and cut into 1-2 mm segments and then plated on corn meal agar amended with pimaricin (5 μg/ml), ampicillin (250 μg/ml), rifampicin (10 μg/ml), pentachloronitrobenzene (50 μg/ml), and benomyl (10 μg/ml) (PARP+B), which is selective for oomycetes (Jeffers and Martin 1986). After 3 days of incubation in the dark at 25℃, colonies were transferred to 10% V8 juice agar and incubated at 25℃ for 2 weeks. Six isolates were identified as Pythium torulosum based on the morphology of sexual and asexual structures following van der Plaats-Niterink's key (van der Plaats-Niterink 1981). On 10% V8 juice agar, the hypha were aseptate and colonies had filamentous sporangia with a dendroid or globose structure. The oogonia were globose or subglobose, laevis, terminal, rarely intercalary, ranging from 12-19 (average 16) μm. Antheridia were mostly sessile or brachypodous, and each oogonium was supplied by 1-2 antheridia cells. Oospores were globose, plerotic, ranging from 9-16 (average 13) μm. For the molecular identification, two molecular targets, the internal transcribed spacer (ITS) region of ribosomal DNA and cytochrome c oxidase subunit II (CoII), were amplified and sequenced using universal primer sets DC6/ITS4 (Cooke et al. 2000) and FM58/FM66 (Villa et al. 2006), respectively for one isolate, "copt". BLAST analyses of a 971 bp ITS segment amplified from copt (GenBank Accession No. MT830918) showed 99.79% identity with a P. torulosum isolate (GenBank Accession No. AY598624.2). For the COⅡ gene of copt, BLAST analyses of a 553 bp segment (GenBank Accession MT843570) showed 98.37% identity with P. torulosum isolate (GenBank Accession No. AB095065.1). Thus, the isolate, copt, was identified as P. torulosum based on morphological characteristics and molecular analysis. To confirm pathogenicity and Koch's postulates, a pathogenicity test was conducted as described by Zhang et al. (2000). Briefly, a 5 mm culture plug from the P. torulosum isolate, copt, was transferred to a 9-cm petri dish containing 20mL 10% V8 juice agar and incubated in the dark at 25℃ for 7 days. The culture was cut into small pieces and mixed with a sterilized soil mix (40% organic peat substrate, 40% perlite, and 20% soil) at a ratio of one petri dish per 100 g soil mix. Ten corn seeds were planted at a depth of 2 cm in a 500-mL pot containing the inoculated soil mix. The control pots were mock inoculated with plain 10% V8 juice agar. Pots were incubated in a greenhouse at temperatures ranging from 21 to 23℃. There were four replications. After 14 days, corn roots brown and rotted were observed, which was similar to those observed in the field and growth chamber. Control plants remained symptomless and healthy. P. torulosum copt was consistently re-isolated from the symptomatic roots. To our knowledge, this is the first report of P. torulosum causing root rot of corn in Northeastern China. Corn is an important crop in Heilongjiang and the occurrence of root rot caused by this pathogen may be a new threat to corn plants. There is a need to develop management measures to control the disease.

Keywords: Pythium; corn; root rot.

The area bordering three 110-ha (270-acre) fields of blighted potatoes (Solanum tuberosum L.) in three northeastern Maine locations was surveyed during the summer of 2004 for the occurrence of late blight on cultivated and noncultivated host plants. Special attention was directed to solanaceous weed species. Hundreds of Solanum sarrachoides Sendt. ex. Mart. (hairy nightshade) plants with numerous leaf lesions and moderate defoliation were seen. The frequency of blighted hairy nightshade approximated the frequency of late blight in the adjoining potato fields. Lesions typically contained extensive, white, superficial mycelia colonizing the abaxial and adaxial leaf surfaces. Samples placed in a moist chamber produced lemon-shaped sporangia. On the basis of morphological characteristics, the pathogen was tentatively identified as Phytophthora infestans (Mont.) de Bary. Isolates were obtained by surface-disinfecting leaf sections collected from two locations for 2 to 3 min in 0.5% NaOCl and placing the sections on rye grain medium amended with antibiotics (100 ppm each of penicillin G, pimaricin, and polymyxin). P. infestans was confirmed after reisolating onto rye-lima bean medium. Pathogenicity was tested on detached potato, tomato, and hairy nightshade leaves; the undersides of all leaflets from replicate plants were inoculated with droplets of swimming zoospores (≥500 zoospores per droplet), the leaves were kept at 17°C and 100% humidity, and the extent of sporulation was evaluated after 4, 6, and 7 days. With eight isolates obtained from S. sarrachoides, Koch's postulates were completed on potato and hairy nightshade. Radial growth responses of these strains on rye grain agar amended with 1, 10, or 100 μg per ml of metalaxyl (Ridomil 2E) yielded 50% effective dose values greater than 100 μg per ml, since percentage growth at the highest fungicide concentration exceeded 50% of the no metalaxyl control. These resistance levels are typical of the metalaxyl-insensitive strains of P. infestans isolated from potatoes in this area in recent years, which were previously found to correlate with metalaxyl resistance in bioassays using potato tissues (1). Eight single-sporangial isolates were homozygous for glucose-6-phosphate isomerase and peptidase (Gpi 100/111/122, Pep 100/100). All eight were A2-mating type and mitochondrial haplotype Ia, characteristics common to the US-8 clonal lineage of P. infestans from potato (2), which may infect a wider host range than the old US-1 clonal lineage. When evaluated on differential hosts, three isolates were tomato race PH-1 and complex potato race R 0,1,2,3,4,9,11. DNA fingerprint analysis with probe RG57 further established that the eight hairy nightshade isolates were identical to each other and to local P. infestans isolates from potato. To our knowledge, this is the first report of infection of S. sarrachoides by P. infestans in Maine. The pathogen was previously isolated from this host during field surveys in southern California in the 1980s in connection with late blight of tomato (4). Hairy nightshade has been shown to be a host for US-1, US-8, and US-11 isolates of P. infestans in a laboratory setting (3). The epidemiological significance of S. sarrachoides as an alternative or overwintering host of P. infestans is currently being assessed. References: (1) K. L. Deahl et al. Am. Potato J. 70:779, 1993. (2) S. B. Goodwin et al. Phytopathology 88:939, 1998. (3) H. W. Platt. Can. J. Plant Pathol. 21:301, 1999. (4) V. G. Vartanian and R. M. Endo Plant Dis. 69:516, 1985.