Effective Management of Citrus Melanose Based on Combination of Ecofriendly Chemicals.

Citrus melanose, caused by the ascomycete fungus Diaporthe citri, is one of the most important diseases in China that affects not only the production but also the quality of citrus. In China, mancozeb is recommended to control melanose disease at the dose of 1.34 g/liter. However, it is widely applied in practice at the dose of 2.66 g/liter or even 4 g/liter, because reduced efficacy of the recommended dose was observed in regions severely damaged by melanose. In this study, some ecofriendly chemicals for melanose management were evaluated. First, the sensitivity to fungicides was screened in the laboratory based on the inhibition of mycelial growth and conidial germination of D. citri. Results showed that both quinone outside inhibitor (QoI) fungicides kresoxim-methyl and trifloxystrobin inhibited conidial germination of D. citri up to 100% at 0.1 µg/ml. The in vivo control efficacy on detached fruit indicated that treatments with elastic nanocopolymer film at 2 g/liter, mancozeb at 1 g/liter, and kresoxim-methyl at 0.1 g/liter significantly inhibited the infection process compared with the control treatment of mineral oil alone. In field trials, the efficacy of kresoxim-methyl at 0.1 g/liter and elastic nanocopolymer film at 2 g/liter mixed with mancozeb at 1 g/liter was equal to that of mancozeb at 2.66 g/liter. The use of mancozeb could be reduced greatly, and the newly developed fungicide combinations are more environmentally friendly due to the low toxicity of both QoI fungicides and elastic nanocopolymer film. The newly developed method with ecofriendly chemicals should play an important role in the management of citrus melanose in the future.

Phylogenetic Analysis and Fungicide Baseline Sensitivities of Monilia mumecola in China.

Monilia mumecola is one of the causal agents of peach brown rot in China. In this study, M. mumecola isolates from different locations and hosts were used to analyze the genetic diversity and to assay the sensitivity to four generally used fungicides: carbendazim, tebuconazole, azoxystrobin, and boscalid. Results showed that isolates from different locations tended to be separated. Interestingly, isolates from different hosts (e.g., peach and apricot) at the same locations generally clustered together, indicating that the M. mumecola isolates may infect different hosts in the same areas. The fungicide sensitivity assay of 93 M. mumecola isolates showed that the average effective concentration for 50% mycelial growth inhibition values for carbendazim, tebuconazole, azoxystrobin, and boscalid were 0.103, 0.034, 0.325, and 0.419 µg/ml, respectively. The sensitivity distributions of the tested isolates to the four fungicides showed continuous unimodal curves, indicating no qualitative shift of resistance. No significant difference of sensitivity to tested fungicides was observed among isolates from either different locations or different hosts.

Detection and Molecular Characterization of Resistance to the Dicarboximide and Benzamide Fungicides in Botrytis cinerea From Tomato in Hubei Province, China.

Altogether, 192 Botrytis cinerea isolates collected from tomato greenhouses at different locations in Hubei Province were evaluated for their sensitivity to fungicides procymidone and zoxamide. The mean effective concentration to cause 50% growth inhibition (EC50) values of procymidone for sensitive and resistant isolates were 0.25 and 3.60 µg/ml, respectively. The frequency of procymidone-resistant (ProR) isolates was 18%, and the highest frequency was recorded in Jingmen. Positive cross-resistance was observed for ProR isolates to other dicarboximide fungicides but not to phenylpyrroles. Significant differences were observed for fitness parameters (i.e., mycelial growth, osmotic sensitivity, and virulence between sensitive and resistant isolates). Amino acid sequence of the Bos1 gene revealed that ProR isolates carried either point mutations at codon 365 (I365S) or a pair of point mutations at codons 369 (Q369P) and 373 (N373S). For zoxamide, the mean EC50 values for sensitive and resistant isolates were 0.22 and 5.32 µg/ml, respectively. Approximately 14% of the isolates were found to be resistant to zoxamide, and the highest frequency of resistance was also observed in Jingmen. There was positive cross-resistance for zoxamide-resistant (ZoxR) isolates to carbendazim. No significant differences were observed for fitness parameters between zoxamide-sensitive and ZoxR isolates. Sequence analysis of the ß-tubulin gene of Botrytis cinerea revealed two previously reported point mutations (E198A and E198K) and one new point mutation (T351I). This new mutation was detected in only those isolates which possessed the E198K but not E198A substitution. This study allows for a better understanding of the resistance development profile in Hubei Province. Results will be useful for the improvement of fungicide resistance management strategies.

Sensitivity of Botrytis cinerea From Nectarine/Cherry in China to Six Fungicides and Characterization of Resistant Isolates.

Botrytis cinerea, the causal agent of gray mold, can result in considerable preharvest and postharvest losses in many economically valuable plant species. Fungicides were widely used to minimize such losses, but fungicide resistances were detected frequently. In the present study, we collected 164 isolates from nectarine and cherry in China and tested the sensitivity to six fungicides. Among the tested isolates, 71 (43.3%) were resistant to azoxystrobin, 14 (8.5%) to cyprodinil, 7 (4.3%) to boscalid, 4 (2.4%) to carbendazim, 1 (0.6%) to iprodione, and no isolates were found to be resistant to fludioxonil. The EC50 value and resistance factor (RF) of resistant isolates were determined. Fitness analysis showed that there were no significant differences between sensitive and resistant isolates for osmotic stress and pathogenicity, while more conidia production was observed for some resistant isolates. Control efficacy of fungicides showed that the resistant isolates could not be controlled efficiently by using corresponding fungicides. The point mutation G143A was detected in the Cyt b gene of the isolates resistant to azoxystrobin, while the point mutation H272R of SdhB gene was confirmed in boscalid-resistant isolates, and mutations E198V/A of TUB2 gene and mutation I365S of BcOs1 occurred in carbendazim-resistant and iprodione-resistant isolates, respectively. These results indicate that the occurrence of fungicide resistance greatly threatens the management of gray mold on stone fruits nectarine and cherry.

Development of a LAMP Method for Detecting SDHI Fungicide Resistance in Botrytis cinerea.

Resistance to succinate dehydrogenase inhibitors (SDHI) in Botrytis cinerea is associated with point mutations in the target gene succinate dehydrogenase subunit B (SdhB). The substitution from histidine to arginine at codon 272 (H272R) is currently the predominant mutation in SDHI-resistant populations in B. cinerea worldwide. In order to monitor the development of resistance to SDHI, a rapid, simple, and efficient method with high specificity to the H272R point mutation was developed based on loop-mediated isothermal amplification (LAMP). To specifically detect the H272R mutation, a set of four primers was designed based on the sequence of SdhB, and the LAMP reaction was optimized. When SYBR Green I was added after reaction, only samples with the H272R mutation showed the color change (from brown to fluorescent yellow), indicating that this set of primers could successfully discriminate the H272R genotype from other genotypes. Specificity and accuracy tests showed that this LAMP assay had high specificity and accuracy. Moreover, the LAMP method was further simplified with fungal mycelia and conidia as the amplification template which could be prepared within 5 min. Due to the low cost, simplicity, high efficiency, and specificity, the developed LAMP assay may contribute to the monitoring of resistance development to SDHI in B. cinerea, especially in field and high-throughput experiments.

Multiple Fungicide Resistance in Botrytis cinerea from Greenhouse Strawberries in Hubei Province, China.

Two hundred and forty isolates of Botrytis cinerea were collected during the early summer of 2012 and 2013 from strawberry greenhouses in 10 locations in Hubei Province and examined for sensitivity to five fungicides, most of which were commonly used to control this fungus. High frequency of resistance to carbendazim (Car, 63.63%) and cyprodinil (Cyp, 42.42%) was detected. Boscalid-resistant (BosR) isolates were detected for the first time in China, whereas no fludioxonil-resistant isolates were identified. Dual resistance to carbendazim and diethofencarb (Die) was also detected. There were six phenotypes of resistance profile (i.e., CarRDieSBosSCypS, CarRDieRBosSCypS, CarRDieSBosSCypR, CarRDieSBosRCypS, CarRDieRBosSCypR, and CarRDieSBosRCypR). CarRDieSBosSCypS and CarRDieSBosSCypR were the most common phenotypes, occurring at eight and seven locations, respectively. After 10 successive transfers on fungicide-free potato dextrose agar, tested resistant isolates retained levels of resistance similar to or comparative with the initial generation, indicating the stability of these resistances. Fitness evaluations based on investigation of mycelial growth, osmotic sensitivity, sporulation in vitro and in vivo, and virulence revealed the uncompromising fitness in resistant isolates, except that decreased virulence was observed in BosR isolates. The molecular basis of carbendazim, diethofencarb, and boscalid resistance was investigated. Results showed that all 13 sequenced carbendazim-resistant isolates harbored the mutation E198V or E198A in the ß-tubulin gene and the five isolates with dual resistance to carbendazim and diethofencarb showed the mutation E198K in the same gene. BosR isolates possessed the H272R mutation in succinate dehydrogenase subunit B gene. The results achieved in this study challenge the current management strategies for B. cinerea, which largely depend on applications of these fungicides.

Fitness and Competitive Ability of Botrytis cinerea Isolates with Resistance to Multiple Chemical Classes of Fungicides.

Resistance to multiple chemical classes of fungicides in Botrytis cinerea isolates from eastern United States strawberry fields is common and strategies to control them are needed. In this study, we compared fitness and competitive ability of eight sensitive isolates (S), eight isolates resistant to five or six chemical classes of fungicides but not to phenylpyrroles (5CCR), and eight isolates resistant to six or seven chemical classes including phenylpyrroles (6CCR/MDR1h). The latter included the MDR1h phenotype due to overexpression of atrB based on Δ497V/L in mrr1. The 6CCR/MDR1h isolates grew more slowly at 4°C on potato dextrose agar, and both 5CCR and 6CCR/MDR1h isolates were hypersensitive to osmotic stress compared with S isolates. In contrast, no differences were found in oxidative sensitivity, aggressiveness, and spore production in vivo, and sclerotia production and viability in vitro. In competition experiments, the 5CCR and 6CCR/MDR1h isolates were both outcompeted by S isolates and 6CCR/MDR1h isolates were outcompeted by 5CCR isolates in the absence of fungicide pressure. Under selective pressure of a fludioxonil/pyraclostrobin rotation, the 6CCR/MDR1h isolates dominated after coinoculation with 5CCR and S isolates. The competitive disadvantage of 5CCR and especially 6CCR/MDR1h isolates suggest that, in the absence of fungicide selection pressure, S isolates may reduce inoculum potential of multifungicide-resistant isolates under field conditions.

Occurrence of Fungicide Resistance in Botrytis cinerea from Greenhouse Tomato in Hubei Province, China.

During the early summer of 2012 and 2013, samples of gray mold were collected from greenhouse tomato at eight locations in Hubei Province, and 221 isolates of Botrytis cinerea were obtained and evaluated for the sensitivity to fungicides carbendazim, diethofencarb, boscalid, fludioxonil, and cyprodinil. Results showed that isolates with resistance to carbendazim and cyprodinil were widespread, whereas isolates with resistance to carbendazim and diethofencarb were found at only two locations. No isolates with resistance to boscalid or fludioxonil were detected. Altogether, four resistant phenotypes were determined (i.e., CarRDieSCypS, CarRDieRCypS, CarRDieSCypR, and CarRDieRCypR). Among them, CarRDieSCypS and CarRDieSCypR were widely distributed, and there was a dominant resistant phenotype at each location. Interestingly, isolates resistant only to cyprodinil were not obtained because the resistance to cyprodinil was always associated with the resistance to carbendazim, demonstrating that a phenotype of multiple fungicide resistance of B. cinerea was more likely to have evolved from previously resistant subpopulations. Stability of resistance to carbendazim, diethofencarb, and cyprodinil was assessed, and the resistance was stable. Fitness tests showed that, as a group, the carbendazim-resistant isolates were not significantly different from sensitive isolates. However, the mycelial growth and virulence of the carbendazim, diethofencarb, and cyprodinil triple-resistant group were significantly lower than the sensitive group, indicating that the triple-resistant isolates suffered from the disability of colonizing the hosts. It should be noted that there was no significant difference for other fitness components (e.g., sporulation or osmotic sensitivity to NaCl), suggesting that the triple-resistant isolates were still competitive in these traits. To investigate the mechanisms of resistance to carbendazim and diethofencarb, partial ß-tubulin genes of 10 carbendazim-resistant isolates and 5 isolates resistant to carbendazim and diethofencarb were sequenced, and all 10 carbendazim-resistant isolates harbored the mutation E198V or E198A. For the 5 isolates resistant to carbendazim and diethofencarb, all of them possessed the mutation E198K, and no other mutations were detected. The location-specific resistance profiles found in this study are crucial in designing proper gray mold management strategies in these areas.

Sensitivity of Colletotrichum Species, Including C. fioriniae and C. nymphaeae, from Peach to Demethylation Inhibitor Fungicides.

Few fungicides are effective against anthracnose, caused by Colletotrichum spp., and emerging resistance makes the search for chemical alternatives more relevant. Isolates of the Colletotrichum acutatum species complex were collected from South Carolina and Georgia peach orchards and phylogenetic analysis of the combined internal transcribed spacer region, glyceraldehyde-3-phosphate dehydrogenase, and ß-tubulin gene sequences separated the isolates into C. nymphaeae and C. fioriniae. The sensitivity of these and three other previously reported Colletotrichum spp. from peach, including C. fructicola, C. siamense, and C. truncatum, to demethylation inhibitor (DMI) fungicides difenoconazole, propiconazole, tebuconazole, metconazole, flutriafol, and fenbuconazole was determined based upon mycelial growth inhibition. C. truncatum was resistant to tebuconazole, metconazole, flutriafol, and fenbuconazole and C. nymphaeae was resistant to flutriafol and fenbuconazole based on 50% effective concentration (EC50) values >100 µg/ml. C. fructicola and C. siamense were sensitive to all DMI fungicides (EC50 values of 0.2 to 13.1 µg/ml). C. fioriniae subgroup 2 isolates were less sensitive to DMI fungicides (EC50 values of 0.5 to 16.2 µg/ml) compared with C. fioriniae subgroup 1 (EC50 values of 0.03 to 2.1 µg/ml). Difenoconazole and propiconazole provided the best control efficacy in vitro to all five species, with EC50 values of 0.2 to 2.7 µg/ml. Tebuconazole and metconazole were effective against all Colletotrichum spp., except for C. truncatum. The strong in vitro activity of some DMI fungicides against Colletotrichum spp. may be exploited for improved anthracnose disease management of peach.

Sensitivity of Monilinia fructicola from Peach Farms in China to Four Fungicides and Characterization of Isolates Resistant to Carbendazim and Azoxystrobin.

Brown rot of peach caused by Monilinia fructicola can cause considerable preharvest and postharvest losses in China. Fungicides are increasingly utilized to minimize such losses. Eighty isolates of M. fructicola were collected from commercial peach orchards located in five provinces in China and the sensitivity to carbendazim, azoxystrobin, tebuconazole, and boscalid was determined. Resistance to carbendazim was detected only in the Yunnan province in 15 of 16 isolates. Characterization of carbendazim-resistant isolates revealed stable resistance, no fitness penalty, and negative cross resistance to diethofencarb. Resistant isolates produced disease symptoms on detached fruit sprayed with label rates of formulated carbendazim and possessed the amino acid mutation E198A in ß-tubulin. Resistance to azoxystrobin was detected in 3 of 10 isolates from Fujian. In contrast to carbendazim resistance, however, azoxystrobin resistance was unstable, associated with a fitness penalty, and not associated with mutations in the target gene cytochrome b. The concentration at which mycelial growth is inhibited 50% (EC50) values of the azoxystrobin-sensitive isolates were 0.02 to 1.94 µg/ml, with a mean value of 0.54 µg/ml. All isolates were sensitive to tebuconazole, with a mean EC50 value of 0.03 µg/ml. The EC50 values for boscalid were 0.01 to 3.85 µg/ml, with a mean value of 1.02 µg/ml. Our results indicate that methyl benzimidazole carbamates (MBCs), quionon outside inhibitors, demethylation inhibitor fungicides, and succinate dehydrogenase inhibitors are likely to be very effective in controlling brown rot in many peach production areas in China, but that resistance to MBCs is emerging.

First Report of Brown Rot of Apricot Caused by Monilia mumecola.

In May 2013, apricot (Prunus armeniaca) fruits covered with grayish, conidial masses were collected from an unknown cultivar in an experimental field of Huazhong Agricultural University, Wuhan, Hubei Province. About 3 to 5% of fruit was infected and affected apricots had tan to white zones of sporulation, which resembled brown rot caused by Monilia species. Conidia were harvested from the surface of the sporulating apricot fruit and spread onto shallow potato dextrose agar (PDA) media (about 2 mm in thickness) using sterile cotton swabs. Conidia were lemon-shaped and mean size was 15.7 (10 to 22.5) × 25 (16.25 to 35) µm. Conidia on PDA were incubated at 23°C for 3 h in darkness, then observed under microscope. More than two germ tubes were produced from each conidium, which was the distinctive trait of Monilia mumecola species (2). Single-spore isolates were obtained and 3 isolates were cultured on PDA in petri dishes. Mycelium grew at an average of 15 mm per day, and the colony showed concentric rings of mycelium with lobbed margins at 23°C in darkness. A 712-bp fragment was PCR amplified from ß-tubulin gene (TUB2) of all the nine isolates investigated indicative of M. mumecola (2). The ribosomal ITS1-5.8S-ITS2 regions of nine isolates were also PCR-amplified from genomic DNA using primers ITS1 and ITS4 and then sequenced (4). ITS sequences were identical to ITS sequences of M. mumecola from China (HQ908786) and Japan (AB125613, AB125614, and AB125620), but only has 98% and 97% identity with the closest species M. laxa (EU042149) and M. fructicola (HQ908789) according to BLAST search in GenBank. Pathogenicity was confirmed by inoculating mycelial plugs of three isolates into six surface-sterilized apricots wounded with a 6-mm diameter sterile cork borer. Control fruit received plain PDA plugs and was incubated in a moist chamber at 23°C with 12 h light/12 h dark. All inoculated fruit developed typical brown rot symptoms with sporulating areas as described above after 3 days of incubation, while control fruits remained healthy. The developing spores on inoculated fruit were re-isolated and confirmed to be M. mumecola. M. mumecola was first isolated from Prunus mume in Japan in 1982 as an unknown Monilia species (3), then identified and the nomenclature was provided in 2004 (1). To our knowledge, this is the first report of M. mumecola on P. armeniaca indicating that M. mumecola has spread to different hosts. References: (1) Y. Harada et al. J. Gen. Plant Pathol. 70:297, 2004. (2) M. J. Hu et al. Plos One, 6(9):e24990, 2011. (3) S. Nakao. Kongetsu-no-noyaku, 1:92, 1992. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

First Report of Brown Rot Caused by Monilia mumecola on Chinese Sour Cherry in Chongqing Municipality, China.

Cherry is widely planted in China, from Liaoning, Beijing, Hebei, Shandong, Zhejiang, Jiangsu, and Anhui provinces (eastern China), to Shaanxi, Sichuan, Chongqing, and Guizhou provinces (western China). The brown rot fungus Monilinia fructigena causes considerable production losses in cherry production in Liaoning Province (3). In May 2013, Chinese sour cherry (Prunus pseudocerasus) cv. Wupi displaying symptoms of brown rot was found in an orchard in Chongqing municipality. Diseased cherry fruit had a brown rot sporulating with grayish, conidial tufts. The fruit later succumbed to the soft rot or shivered and became a mummy. Single-spore isolations on PDA resulted in colonies with concentric rings of pigmented mycelium with lobbed margins. Conidia were broadly ellipsoid to subglobose, occasionally even globose, with an average size of 16 × 12.7 µm. Multiple germ tubes were produced from each conidium, a germination pattern unique to Monilia mumecola (1,2,4). The pathogen identity was confirmed by multiplex PCR as described by Hu et al. (2). The PCR resulted in a 712-bp amplicon, which is diagnostic of M. mumecola. Further sequencing of the internal transcribed spacer (ITS) region 1 and 2 and 5.8S gene further indicated 100% identity with that of M. mumecola isolates from China (Accession No. HQ908786) and from Japan (AB125613, AB125614, and AB125620). Koch's postulates were confirmed by inoculating mature cherry fruit with mycelia plugs. Inoculated fruit were placed in a sterilized moist chamber, and incubated at 22°C with 12 h light/dark cycle. Inoculated fruit developed typical brown rot symptoms only 2 days after inoculation, while the control fruit, inoculated with a sterile PDA plug, remained healthy. The pathogen isolated from inoculated symptomatic fruit was confirmed to be M. mumecola based on morphological characteristics and germination pattern. It should be noted that the conidia on inoculated fruit showed an average size of 20 × 15.3 µm, significantly bigger than that of from PDA, and most produced more than three germ tubes. The inoculation experiments were performed in triplicates. M. mumecola was first reported as the causal agent of brown rot of mume in Japan in 2004 (1). Later studies demonstrated that it is also pathogen on other stone fruits, e.g., peach, nectarine (2), and apricot (4). To our knowledge, this is the first report of cherry brown fruit rot caused by M. mumecola, and the first report of M. mumecola in Chongqing municipality. References: (1) Y. Harada et al. J. Gen. Plant Pathol. 70:297, 2004. (2) M. J. Hu et al. Plos One 6(9): e24990, 2011. (3) Z. H. Liu et al. J. Fruit Sci. 29:423, 2012. (4) L. F. Yin et al. Plant Dis. 98:694, 2014.

First Report of Peach Brown Rot Caused by Monilinia fructicola in Central and Western China.

Brown rot of peach (Prunus persica) in China has been reported to be caused by at least three Monilinia species (1). In the present study, peaches with symptoms resembling brown rot caused by Monilinia species were collected from commercial orchards in the northwestern province of Gansu in August 2010, the southwestern province of Yunnan in July 2011, and in the central province of Hubei in July 2012. Affected fruit showed the typical symptoms of brown rot with zones of sporulation. Fungal isolates were single-spored and cultured on potato dextrose agar (PDA). Colonies showed grayness with concentric rings of sporulation after incubation at 25°C in the dark. Mean mycelial growth of isolates YHC11-1a and YHC11-2a from Yunnan, GTC10-1a and GTC10-2a from Gansu, and HWC12-14a and HWC12-23a from Hubei, was 4.6 ± 0.4 and 7.5 ± 0.7 cm after 3 and 5 days incubation, respectively. Conidia were lemon shaped and formed in branched monilioid chains, and the mean size was 9.3 (6.7 to 11.5) × 12.5 (7.9 to 17.8) µm, which was consistent with the characteristics of Monilinia fructicola (1,2). The species identification was confirmed by sequencing of the ribosomal ITS sequences. The ribosomal ITS1-5.8S-ITS2 region was amplified from each of the six isolates using primers ITS1 and ITS4 (3). Results indicated that the ITS sequences of these isolates were identical and showed the highest similarity (100%) with M. fructicola ITS sequences from isolates collected in China (GenBank Accession Nos. HQ893748, FJ515894, and AM887528), Slovenia (GU967379), Italy (FJ411109), and Spain (EF207423). The pathogen was also confirmed to be M. fructicola based on the detection of an M. fructicola- specific band (534 bp) using a PCR-based molecular tool developed for distinguishing Chinese Monilinia species affecting peach (1). Pathogenicity was tested on surface-sterilized, mature peaches (Shui Mi Tao) with representative isolates. Fruits were holed at three equidistant positions to a depth of 5 mm using a sterile cork borer. Mycelial plugs (5 mm in diameter) from the periphery of a 4-day-old colony of each isolate were placed upside down into each hole, control fruits received water agar. After 3 days of incubation at 22°C in a moist chamber, inoculated fruits developed typical brown rot symptoms while control fruits remained healthy. Pathogens from the inoculated fruit were confirmed to be M. fructicola based on morphological characteristics. To our knowledge, this is the first report of occurrence of M. fructicola in Gansu, Yunnan, and Hubei provinces, thousands of kilometers away from eastern China where occurrence of peach brown rot caused by M. fructicola has been confirmed (2,4). The results indicated the further geographical spread of the M. fructicola in China. References: (1) M. J. Hu et al. Plos One 6(9):e24990, 2011. (2) M. J. Hu et al. Plant Dis. 95:225, 2011. (3) T. J. White et al. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Academic Press, San Diego, 1990. (4) X. Q. Zhu et al. Plant Pathol. 54:575, 2005.

First Report of Brown Rot of Apple Caused by Monilinia fructicola in Germany.

Monilinia fructicola (G. Wint.) Honey is a causal agent of brown rot of stone fruits but may also affect pome fruits. M. fructicola is common in North America, Oceania, and South America as well as in Asia, but it is listed as a quarantine pathogen in Europe (3). Since its first discovery in Europe in 2001 (France), it has been reported in Spain, Slovenia, Italy, and Switzerland. Recently, the fungus was also detected in orchards of blackberries and plums in the State of Baden-Württemberg, Germany (4). In July 2010, apples (Malus domestica Borkh.) of the cultivar Jonagold were found in a residential backyard in Fronhausen an der Lahn located in the State of Hessen, Germany with symptoms resembling brown rot caused by Monilinia species. Affected apples were at or near maturity with brown decay that had spread throughout the fruits. On the surface of the decaying apples was tan to white zones of sporulation. Upon isolation, the mycelium grew at a linear rate of 9.2 mm per day at 22°C on potato dextrose agar forming branched, monilioid chains of grayish colonies with concentric rings and little sporulation. The lemon-shaped spores had an average size of 14 × 9 µm, a shape and size consistent with M. fructicola. The ribosomal ITS1-5.8S-ITS2 region was PCR-amplified from genomic DNA obtained from mycelium using primers ITS1 and ITS4. A BLAST search in GenBank revealed highest similarity (99%) to M. fructicola sequences from isolates collected in China, Italy, and Slovenia (GenBank Accession Nos. FJ515894.1, FJ411109.1, GU967379.1). The M. fructicola sequence from the apple isolate was submitted to GenBank (Accession No. JF325841). The pathogen was also identified to the species level and confirmed to be M. fructicola using two novel PCR techniques based on cytochrome b sequences (1,2). Pathogenicity was confirmed by inoculating three surface-sterilized, mature apples cv. Gala with a conidial suspension (105 spores/ml) of the apple isolate. Fruit were stab inoculated at three equidistant points to a depth of 10 mm using a sterile needle. A 30-µl droplet was placed on each wound; control fruit received sterile water without conidia. After 5 days of incubation at room temperature in air-tight plastic bags, the inoculated fruits developed typical brown rot symptoms with sporulating areas (as described above). The developing spores on inoculated fruit were confirmed to be M. fructicola. All control fruits remained healthy. To our knowledge, this is the first report of M. fructicola on apple in Germany and more indication of further geographical spread of the quarantine disease in Germany. References: (1) J.-M. Hily et al. Pest Manag. Sci. Online publication. doi 10.1002/ps.2074, 2011. (2) S. Miessner and G. Stammler. J. Plant Dis. Prot. 117:162, 2010. (3) OEPP/EPPO. EPPO A2 list of pests recommended for regulation as quarantine pests. Version 2009-09. Retrieved from http://www.eppo.org/QUARANTINE/listA2.htm , September 22, 2010. (4) OEPP/EPPO. Reporting Service. No. 1, January 2010. Retrieved from http://archives.eppo.org/EPPOReporting/2010/Rse-1001.pdf , September 22, 2010.

First Report of Brown Rot of Peach Caused by Monilinia fructicola in Southeastern China.

In 2009 and 2010, peaches (Prunus persica) with brown rot symptoms were collected from Zhejiang Plant Protection State Research Farm and a commercial orchard in Fujian Province in southeastern China. Affected fruit showed brown decay with zones of sporulation. Single-spore isolates from the diseased fruit were cultured on potato dextrose agar. After incubation at 25°C in the dark for 5 days, colonies were gray with concentric rings of sporulation. Mean mycelial growth of isolates MZ09-2a from Zhejiang Province and 0907-a from Fujian Province was 4.46 ± 0.58 and 7.05 ± 0.81 cm after 4 and 7 days of incubation, respectively. Lemon-shaped conidia were formed in branched, monilioid chains and mean size was 14.6 (9.6 to 21.6) × 10.3 (7.2 to 13.2) µm. Mean conidial germination was 97 ± 1% with one straight germ tube per conidium. These characteristics were consistent with descriptions of Monilinia fructicola (G. Wint.) Honey (3). Morphology-based species identification was confirmed by sequencing and analysis of ribosomal internal transcribed spacer (ITS) sequences. A 496-bp fragment including ITS 1 and 2 and the gene encoding the 5.8S small subunit of the ribosomal RNA from isolates MZ09-2a and 0907-a was amplified using the universal primer pair ITS1/ITS4 (4) and sequenced. Nucleotide sequences of both isolates were identical. Blast searches of the ITS sequences in GenBank showed the highest similarity (100%) with sequences of M. fructicola isolates from China (FJ515894), Italy (FJ411109), and Spain (EF207423). The isolates were also identified as M. fructicola using the Monilinia spp. PCR detection protocol based on sequence-characterized amplification region marker DNA sequences (2). Pathogenicity was confirmed by inoculating surface-sterilized, mature cv. Zhonghua 2 peaches with mycelial plugs of representative isolates. Fruit was stabbed at two points with a 5-mm-diameter sterile cork borer, mycelial plugs (5 mm in diameter) were removed from the periphery of a 4-day-old colony of each isolate and placed upside down into each wound; control fruit received water agar. Inoculated fruit developed typical brown rot symptoms with sporulating fungi while control fruit remained healthy after 3 days of incubation at 22°C in a moist chamber. Pathogens were reisolated from the inoculated fruit and confirmed to be M. fructicola on the basis of morphological characteristics. To our knowledge, this is the first report of M. fructicola in Zhejiang and Fujian provinces. Both provinces are located more than 1,000 km south of Beijing, Hebei, and Shandong provinces, where M. fructicola had been reported previously (1). References: (1) J. Y. Fan et al. Acta Phytophylacica Sin. (in Chinese) 34:289, 2007. (2) I. Gell et al. J. Appl. Microbiol. 103:2629, 2007. (3) G. C. M. van Leeuwen and H. A. van Kesteren. Can. J. Bot. 76:2041, 1998. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds., Academic Press, San Diego, 1990.

Protective effects of 20-hydroxyecdysone on CoCl(2)-induced cell injury in PC12 cells.

20-Hydroxyecdysone, which is found in the rhizomes, roots and the stems of many plants, is an ecdysteroid hormone that regulates molting in insects. We have previously shown that 20-Hydroxyecdysone could alleviate neurological deficits induced by subarachnoid hemorrhage in rabbits. Thus, we hypothesized that 20-Hydroxyecdysone might protect neurons against hypoxic-ischemic injury. In present study, the effects of 20-Hydroxyecdysone on cobalt chloride (CoCl(2))-induced cellular injury in PC12 cells was investigated. The incubation of PC12 cells with CoCl(2) reduced the cell viability, increased the rate of apoptosis. However, when cells were treated with 20-Hydroxyecdysone before or after CoCl(2) exposure, the CoCl(2)-induced cellular injuries were significantly ameliorated. In addition, 20-Hydroxyecdysone dramatically reduced the CoCl(2)-induced production of reactive oxygen species (ROS), decreased the depolarization of the mitochondrial membrane, inhibited the release of cytochrome c into the cytosol and increased the Bax/Bcl-2 ratio. Furthermore, 20-Hydroxyecdysone eliminated the CoCl(2)-induced activation of caspase-3. Taken together, these results indicate that 20-Hydroxyecdysone may protect PC12 cells against CoCl(2)-induced cell injury by inhibiting ROS production and modulating components of the mitochondrial apoptosis pathway. Based on our results, 20-Hydroxyecdysone may be a potential candidate for intervention in hypoxic-ischemic brain injuries such as stroke.

Neuronal nitric oxide synthase-derived nitric oxide inhibits neurogenesis in the adult dentate gyrus by down-regulating cyclic AMP response element binding protein phosphorylation.

Neuronal nitric oxide synthase, the major nitric oxide synthase isoform in the mammalian brain, is implicated in some developmental processes, including neuronal survival, precursor proliferation and differentiation. However, reports about the role of neuronal nitric oxide synthase in neurogenesis in the adult dentate gyrus are conflicting. Here we show that 5-bromodeoxyuridine-labeled dividing progenitor cells in the dentate gyrus were significantly increased in mice receiving 7-nitroindazole, a selective neuronal nitric oxide synthase inhibitor, and in null mutant mice lacking neuronal nitric oxide synthase gene (nNOS-/-) 6 h and 4 weeks after 5-bromodeoxyuridine incorporation. The increase in 5-bromodeoxyuridine positive cells in 7-nitroindazole-treated mice was accompanied by activation of cyclic AMP response element binding protein phosphorylation in the dentate gyrus. Pretreatment with N-methyl-D-aspartate receptor antagonist MK-801 fully abolished the effects of 7-nitroindazole on neurogenesis and cyclic AMP response element binding protein phosphorylation. Furthermore, neuronal nitric oxide synthase inhibition significantly enhanced the survival of newborn cells and the number of 5-bromodeoxyuridine positive/NeuN positive cells in the dentate gyrus. These results indicate that neuronal nitric oxide synthase-derived nitric oxide suppresses neurogenesis in the adult dentate gyrus, in which N-methyl-D-aspartate receptor functions and cyclic AMP response element binding protein phosphorylation may be involved.

Identification of Magnaporthe oryzae Avirulence Genes to Three Rice Blast Resistance Genes.

The segregation of avirulence/virulence was studied in 115 F1 progeny isolates of Magnaporthe oryzae from a cross of two field isolates on three Japanese race-differential rice cultivars Kanto 51, Fukunishiki, and Toride 1. The χ2 tests of goodness-of-fit for a 1:1 ratio indicated that avirulence on cvs. Kanto 51, Fukunishiki, and Toride 1 was under monogenic control. The relationship between the avirulence (Avr) gene in the parental isolate and the Avr gene in the standard isolate was investigated by using 100 lines each of three F3 families from the crosses of the rice cultivars Norin 3/Kanto 51, AK61/Fukunishiki, and Norin 3/Toride 1, respectively. Based on the resistant reactions of the F3 rice lines to the parental isolates and the standard isolates harboring three known Avr genes, three genetically independent Avr genes, AvrPik, AvrPiz, and AvrPiz-t, were identified. The three identified Avr genes were mapped using random amplified polymorphic DNA (RAPD) analysis, and a partial linkage map was constructed with 17 RAPD markers closely linked to the Avr genes. Twelve markers and AvrPik, three markers and AvrPiz, and two markers and AvrPiz-t, as well as mating locus MAT1, constructed linkage groups A, B, and C, respectively.

Pharmacokinetic and myocardial enzyme profiles of two administration routes of epirubicin in breast cancer patients.

To evaluate the changes in myocardial enzymes and plasma epirubicin concentration following administration by micro-pump (MP) and intravenous drip (ID) in breast cancer patients.11 self-controlled breast cancer patients were recruited for a trial with epirubicin administration by MP for 48 h and by ID for 1 h during 2 cycles of treatment. Plasma concentration of epirubicin at different time points was determined using LC-MS/MS. The levels of myocardial enzymes before and after chemotherapy were compared. Another group of patients receiving epirubicin by ID (n=4) or MP (n=9) were monitored for 4 months.8 patients completed the self-controlled study. The peak concentration of epirubicin in the MP group and the ID group were 21.84±18.85 ng/mL and 294.80±225.54 ng/mL, respectively. The MP group had a longer duration (54~60 h) of plasma concentration of epirubicin not less than 10 ng/mL than that of the ID group (8~14 h). There was significant difference for the alteration of myocardial enzymes before and after chemotherapy (p<0.05) in the ID group, whereas the MP group showed no significant difference (p>0.05). The increased range of myocardial enzymes after chemotherapy in the ID group was larger than that of the MP group and the difference was significant (p<0.05). There is an increased cardiotoxicity in patients receiving epirubicin by ID during the 4-month trial.Administration of epirubicin by MP maintained an effective drug concentration for a longer period of time than by ID. The higher peak plasma concentration observed following epirubicin administration by ID may lead to cardiac toxicity.

[Intraoperative hemodynamics of pyrolytic carbon cambered bileaflet valve replacement in animals].

Pyrolytic carbon cambered bileaflet valve developed by Chengdu University of Technology and Sciences was evaluated for its hemodynamics. The curved surface of the valve leaflet is cylindrical. Under anaesthesia, cardiopulmonary bypass and chemical cardioplegia, a cambered bileaflet valve (i.d. = 16 mm) was replaced at mitral position in each of 12 goats. Before valve replacement, mitral flow rate (MFR) was 74.9 +/- 12.3 ml.sec-1, average valvular pressure drop delta P) 0.56 +/- 0.17 kPa (4.2 +/- 1.3 mmHg) and effective opening area (EOA) of the valve 1.20 +/- 0.18 cm2. After valve replacement, corresponding hemodynamic parameters were 49.3 +/- 12.7 ml.sec-1, 0.46 +/- 0.17 kPa (3.8 +/- 1.3 mmHg) and 0.81 +/- 0.12 cm2 respectively. In our previous report on yak pericardiac heart valve (i.d. = 20 mm) in goats, corresponding hemodynamic parameters after valve replacement were 45.6 +/- 11.8 ml.sec-1, 0.86 +/- 0.32 kpa (6.4 +/- 2.4 mmHg) and 0.65 +/- 0.26 cm2. With comparable mitral flow rate and heart rate (113.6 +/- 19.6 min-1 vs 119.1 +/- 17.1 min-1), the average valvular pressure drop of pyrolytic carbon cambered bileaflet valve is significantly smaller than that of the yak pericardiac valve (P less than 0.001), and the effective opening area is marginally larger than that of the yak pericardiac valve (0.05 less than P less than 0.1). If the same-sized valves of this two kinds are used, the hemodynamic parameter of cambered bileaflet valve would be better than those of yak pericardiac valve. Since the latter valve has been successfully used in patients, the present valve might be applicable in clinical situation.