Root rot of spinach caused by Pythium myriotylum and P. aphanidermatum in Taiwan.

Spinach (Spinacia oleracea) is a commonly used green vegetable. During September and October in both 2022 and 2023, a vegetable nursery company located among paddy rice fields in Taichung City, Taiwan, reported significant failures in spinach seedling production in net-houses with mean outdoor temperatures of 28.7℃. Abnormal growth was observed in approximately 30% of the spinach seedlings in each batch (n = 2,000 to 3,000), with aboveground tissues showing stunting, yellowing, and wilt, and underground tissues displaying root rot. The symptoms resembled the spinach damping-off documented in Taiwan in extension articles but which lacked complete pathogen identification. A total of 110 plants from two batches were used for pathogen isolation by placing roots on water agar incubated at 25℃ or were examined for the presence of oospores in diseased roots. Eighty-one percent of these plants were associated with Pythium. Nine Pythium isolates were used in subsequent analyses. Genomic DNA from these isolates was subjected to amplification of ITS, ß-tubulin gene (TUB2), and cytochrome C oxidase subunit Ⅱ (COXII) gene with primer pairs ITS1 / ITS4, BT5 / BT6, and FM58 / FM66 (Villa et al. 2006). Sequences of ITS (PP209187-PP209195), TUB2 (PP212864-PP212872), and COXII (PP212855-PP212863) were deposited in GenBank. Four isolates (sp01, sp02, sp03, and sp04) were 100% identical to the neotype strain (CBS 118.80) of Pythium aphanidermatum (Edson) Fitzp. for the ITS (761 bp), TUB2 (583 bp), and COXII (547 bp). Five isolates (2sp, 3sp, ND2-4sp, D3-4sp, and ND3-3sp) were 99.87%, 100%, and 99% identical to the reference strain (CBS 254.70) of Pythium myriotylum Drechsler for the ITS (762 bp), TUB2 (602 bp), and COXII (556 bp), respectively. Phylogenetic analysis of Pythium isolates inferred from concatenated sequences of the three genes (LéVesque and De Cock 2004; Villa et al. 2006) revealed that the same four isolates grouped with the neotype strain of P. aphanidermatum, and the five isolates clustered with the reference strain of P. myriotylum, each with a 100% bootstrap support. Morphological features of isolates ND3-3sp and sp01 were used for identification. Isolate ND3-3sp produced inflated lobulate sporangia and aplerotic and smooth oospores (16.3 to 25.1 um; n = 30) attached with three to five antheridia, consistent with identification as P. myriotylum. Isolate sp01 produced inflated lobulate sporangia and aplerotic and smooth oospores (17.0 to 24.0 um; n= 30) attached with a single intercalary antheridium, agreeing with the morphology of P. aphanidermatum (Van der Plaats-Niterink 1981). To investigate the pathogenicity of the nine Pythium isolates on spinach, 20 mycelial agar discs (4 mm in diameter) from a 2-day-old V8 culture of each isolate were used to induce sporangia and zoospores in 20 ml sterilized water at 25℃ with a 12 h light / dark regime. A 1.5 ml zoospore suspension (6 × 103 zoospores / ml) was dropped into BVB growth substrate of two spinach seedlings in 2-week-old at 25℃ with 12 h light / dark regime, resulting in symptoms resembling those observed in commercial nurseries at 7 days post-inoculation (dpi). Each Pythium isolate inoculated 20 seedlings in 10 cells of a planting tray. At 14 dpi, disease incidences were 95 to 100% for P. myriotylum isolates and 60 to 85% for P. aphanidermatum isolates, while control plants treated with water showed no symptoms. Re-isolated pathogens from the inoculated plants were morphologically identical to the inoculated isolates, completing Koch's postulates. Results of the pathogenicity assay, along with molecular and morphological identification, conclude that the root rot of spinach was caused by P. myriotylum and P. aphanidermatum. The two oomycetes were not formally documented to cause spinach diseases in Taiwan. Although P. myriotylum has been isolated from spinach (Wang et al. 2003), its pathogenicity to spinach was not documented worldwide. Root rot of spinach caused by P. aphanidermatum has been reported in the United States (Bates and Stanghellini 1984), Korea (Cho and Shin 2004), and Italy (Garibaldi et al. 2015). These pathogens thrive in humid and hot weather (Littrell and McCarter, 1970). Producing spinach in cooler weather or in a temperature-controlled environment may help prevent severe occurrence of the disease.

Development of Specific Primers for Fusarium oxysporum Formae Speciales rapae and matthiolae with an Integrated Multiplex PCR for Distinguishing Four Formae Speciales on Brassicaceae.

There are four formae speciales of Fusarium oxysporum responsible for causing yellows of Brassicaceae. Because of crossbreeding among crops, the host ranges of these formae speciales often overlap, making pathogen identification a challenging task. Among these formae speciales, F. oxysporum f. sp. rapae and F. oxysporum f. sp. matthiolae still lack specific primers for pathogen identification. To address this problem, we targeted the secreted in xylem (SIX) genes, known as specific effectors of pathogenic F. oxysporum, for primer design. Through sequence comparison with other formae speciales, we successfully designed specific primers for F. oxysporum f. sp. rapae and F. oxysporum f. sp. matthiolae on SIX14 and SIX9, respectively. Both primer pairs exhibited high specificity in detecting F. oxysporum f. sp. rapae or F. oxysporum f. sp. matthiolae, distinguishing them from 20 nontarget formae speciales of F. oxysporum, five species of phytopathogenic Fusarium, and four other common pathogenic fungi affecting cruciferous plants. Moreover, the effectiveness of these specific primers was validated by detecting the pathogens in infected plants. To further enhance the identification process of the four formae speciales, we combined the two newly designed specific primer pairs with two previously published primer pairs, enabling the establishment of a multiplex PCR method that can accurately distinguish all four formae speciales of F. oxysporum responsible for causing yellows in cruciferous plants in a single reaction.

First report of powdery mildew on Euonymus japonicus caused by Erysiphe euonymicola in Taiwan.

The Japanese spindle (Euonymus japonicus Thunb.) is commonly used as an ornamental hedge plant in Taiwan. In March 2020, a severe powdery mildew disease was observed on E. japonicus surrounding a city park spanning six hectares in Taichung city, Taiwan. Around 90% of the plants showed symptoms on the leaves and pedicels of young shoots. Similar symptoms were observed in other districts of Taichung city and Taipei city between March to June in subsequent years. Initial signs of infection manifest as circular chlorotic spots on the leaves, which are subsequently covered by white mycelia on either the upper or lower surfaces of the spots. In severe cases, both sides of the leaves become entirely covered by dense mycelia. Hyphal appressoria were solitary or in opposite paired, lobed to multilobed. Conidiophores grow erectly from the hyphae, consist of 2-3 cylindrical cells, 38.9 to 78.6 × 6.31 to 8.28 µm (n = 30). Foot cells are usually straight or slightly flexuous, 23.6 to 43.2 µm (n = 30), followed by 1 to 2 shorter cells. Ellipsoidal conidia are produced singly on the conidiophores, 24.1 to 36.3 × 10.6 to 14.97 µm (n = 30), without fibrosin bodies. Germ tubes are mostly subterminal, sometimes terminal, occasionally exhibiting a longitudinal pattern. Chasmothecia were not observed. These morphological characteristics correspond to the description of Erysiphe euonymicola U. Braun (Braun and Cook 2012), one of the Erysiphe species reported on E. japonicus. Genomic DNA was extracted from seven isolates obtained from different plants in the affected regions. The internal transcribed spacer (ITS) and 28S large subunit (LSU) of rDNA sequences (ITS accession nos.: OR073423-OR073429; LSU accession nos.: OR073448-OR073454) were amplified and sequenced using primer sets PMITS-1 / PMITS-2 (Cunnington et al. 2003) and NLP2 / PRM2 (Bradshaw and Tobin 2020), respectively. The resulting sequences exhibited identities ranging from 99.1 to 100% in ITS and 100% in LSU when compared to the corresponding sequences of E. euonymicola MUMH 133 (ITS: AB250228; LSU: AB250230) (Limkaisang et al. 2006). Phylogenetic analysis based on the concatenated sequences of ITS and LSU clustered the seven isolates within the same clade as three E. euonymicola isolates (MUMH 133, MUMH 6999 and MUMH 7012). Pathogenicity assays were conducted on one-meter tall E. japonicus plants by gently smearing infected leaves on all leaves of four healthy plants. Four uninoculated plants were used as control. All eight assayed plants were enclosed in plastic bags to maintain high humidity at 28 ± 2°C for 3 days. Chlorotic spots began to appear on leaves younger than one month old at 7 days post inoculation (dpi). By 28 dpi, all inoculated plants showed symptoms. Spots expanded or merged and formed a dense mycelial layer on leaves younger than three months, while mature dark green leaves were asymptomatic. No symptoms were observed on any leaves of the control plants. The morphological characteristics and sequences of ITS and LSU of the pathogen from the inoculated plants matched the above information. Based on these findings, E. euonymicola was identified as the causal agent of powdery mildew on E. japonicus, representing the first documented report of this disease in Taiwan. A voucher specimen TNM F0037001 (isolate EPM-1) was deposited in the National Museum of Natural Science, Taiwan. The pathogen has been frequently reported in recent years and significantly impacts the ornamental value of Euonymus spp. (Abbasi and Braun 2020; Lee et al. 2015; Li et al. 2011; Pei et al. 2022). This report also provides an evidence of an ongoing outbreak of the pathogen.

Host Invasion Type Is a Phylogenetically Conserved Characteristic of Cephaleuros.

Cephaleuros species cause algal spot diseases, also known as red rust diseases, on many plants, including fruit crops. Most algal species are defined based on their morphological characteristics. Recent phylogenetic studies of Cephaleuros species showed that morphological determination was not congruent with phylogeny. Our study examined the phylogenetic congruence of the host invasion types (or growth habits), which are the most critical characteristics in the taxonomy of Cephaleuros. To ensure that host invasion types and phylogenetic characteristics could be inferred from the same isolate, host invasion types were assessed using microanatomical observation, and rRNA sequences from the same algal spot and/or the derived algal cultures were compared. Host invasion types were found to be conserved classification traits and were congruent with Cephaleuros phylogeny. The results also indicated that more than one Cephaleuros species commonly grew on the same leaf or, in a few cases, the same algal spot, suggesting that identification using different algal spots could result in misidentification. The Cephaleuros isolates were separated into two species complexes by host invasion types: the C. virescens species complex (CVSC) with subcuticular host invasion type and the C. parasiticus species complex (CPSC) with intercellular host invasion type. Molecular phylogenetic analysis indicated that Cephaleuros isolates clustered into 14 clades of CVSC and three clades of CPSC. This study also identified 16 and eight new hosts of CVSC and CPSC in Taiwan, respectively.

First report of Pectobacterium carotovorum and Pectobacterium brasiliense causing bacterial soft rot of bok choy in Taiwan.

Bok choy (Brassica rapa var. chinensis) is one of the most popular leafy green vegetables in Asia (Wang et al. 2019; Zhang et al. 2014). In May 2022, disease resembling bacterial soft rot was observed in a commercial greenhouse located in Xiluo, Yunlin County, Taiwan. Affected plants exhibited maceration, primarily close to the base of the plants (Fig. S1). Almost all bok choy plants (about 1,800 plants in total) on site were symptomatic. Macerated tissues were collected from six plants. The samples were homogenized in 10 mM MgCl2 and bacteria were isolated on nutrient agar (NA) by streak plating. After 1 day of culturing at 28°C, creamy-white, round colonies were consistently grown on all the plates, and six strains (Br1 to Br6) were obtained; each isolated from a different plant. The strains were able to ferment glucose and induced maceration on potato tuber slices (Schaad et al. 2001) but could not produce indigoidine on NGM medium (NA added with glycerol and MnCl2; Lee and Yu 2006). The DNA samples of these strains were tested with Pectobacterium-specific primers Y1 and Y2 (Darrasse et al. 1994) and all samples produced the expected amplicon. To identify the isolated pathogens, 1,592-bp sequences concatenated from fragments of the leuS (452 bp), dnaX (492 bp), and recA (648 bp) genes (GenBank accession nos. OP360013-OP360021) were obtained for each strain as previously described (Portier et al. 2019). Three genotypes were detected, the sequences of strains Br1, Br2, Br4, and Br5 were identical, while strains Br3 and Br6 each belong to a different genotype. The sequence identity between Br3 and Br6 was 98.2%. The concatenated sequences (dnaX-leuS-recA), along with those of type strains from known Pectobacterium species, were subjected to maximum likelihood analysis. The reconstructed trees showed that strains Br1, Br2, Br4, and Br5 grouped with P. carotovorum CFBP2046T (Fig. S2); the sequence identity between the isolated strains and the type strain was 98.7%. Strains Br3 and Br6 clustered with P. brasiliense CFBP6617T (Fig S2); the sequence identity between CFBP6617T and Br3 and Br6 were 97.5% and 98.4%, respectively. The six strains were inoculated onto 55-day-old bok choy plants using previously described prick inoculation methods (Wei et al. 2019). Autoclaved toothpicks, each carrying 9.3 x 106- 5.6 x 107 cfu of bacteria, were used to inoculate the base of plant leaves. All six strains were tested, and each strain had three replicates. Plants in the control group were stabbed with bacteria-free toothpicks. The plants were enclosed in clear plastic bags during the assay to maintain humidity and kept in a growth chamber (27/25°C day/night; 14-h photoperiod). After 1 d, all inoculated plants produced soft rot symptoms resembling those observed in the sampling site. No noticeable differences were observed among symptoms produced by different strains. The controls were symptomless. One strain was re-isolated from each treatment group and their identity were confirmed by sequencing the dnaX gene. All re-isolated strains shared the same sequences with those of the original strains tested. This is the first report of P. brasiliense and P. carotovorum causing bacterial soft rot of bok choy in Taiwan. Importantly, the findings showed that different Pectobacterium species and genotypes could induce symptoms on a crop in the same facility at the same time, highlighting the potential complexity of interactions among different soft rot bacteria in the environment.

First Report of Mango Leaf Blotch Caused by Pseudoplagiostoma mangiferae in Taiwan.

Mango (Mangifera indica L.) is an economically important tropical fruit in southern Taiwan. In February 2019, new leaf blotches distinct from anthracnose lesions were noticed on mango leaves in Meinong, Kaohsiung (N22°54'43.7" E120°32'59.3"). Symptoms were circular to irregular lesions with easily torn centers and were cream to light brown with dark brown margin on both leaf surfaces. Similar symptoms were observed on mango leaves in Yujing, Tainan (N23°07'31.3" E120°27'18.2") in July of the same year. We surveyed the disease incidence on 60 mango trees consisting of three cultivars, 'Irwin', 'Yu-win No.6' and a native cultivar in a commercial farm by randomly examining five shoots of each tree. The disease incidences of 'Irwin', the native cultivar and 'Yu-win No.6' were 25%, 37% and 73%, respectively. Diseased tissues from the two locations were surface sterilized and incubated on potato dextrose agar (PDA) for pathogen isolation. Seven isolates (Mgk3, TMg2-2.2, TMg3-1.2, TMg3-2.1, TMg4-1, TMg6-3, and TMg8-1.1) from different locations and cultivars were selected for further study. Pycnidia were produced on 7-day-old PDA cultures. Conidiogenous cells were hyaline and short cylindrical phialides. Conidia were hyaline, aseptate, thick-walled, 20-24 × 11-16 µm, and subcircular to ellipsoid with 1-2 large oil droplets and a markedly flat protruding hilum at the base. These morphological features presenting in the seven isolates were identified as a member of Pseudoplagiostoma (Cheewangkoon et al. 2010). Pathogenicity assays were conducted by the point inoculation method on 10 intact young leaves growing on 'Irwin' mango plants in a greenhouse at 20-25℃. Each leaf was inoculated at six points on the abaxial surface with point inoculation. Each point was inoculated with a 10 µl conidial suspension (106 conidia/ml) of isolate TMg 8-1.1. Sterilized water was used as control. Shoots with inoculated leaves were covered with translucent plastic bags for 2 days. At 7 days post-inoculation (dpi), 70% of conidia inoculated points (n = 60) displayed symptoms resembling the field symptoms, but sterilized water inoculated points (n = 60) did not. The six other isolates were inoculated on detached leaves by the point inoculation method. All inoculated leaves were maintained in humid containers at 25℃ with 12 h light/dark regime, and displayed similar lesions at 7 dpi. Fungi re-isolated from the symptomatic leaves showed the same morphological characteristics observed in the Pseudoplagiostoma originally isolated from diseased tissues. Internal transcribed spacer (ITS), TUB2 and LSU gene sequences of the seven isolates (ITS accession nos.: MN818659 to MN818665; TUB2 accession nos.: MW415921 to MW415927; LSU accession nos.: MN876849 to MN876855) were amplified with primer sets ITS4/V9G, T1/Bt-2b and LSU1Fd/LR5, respectively, and used for molecular identification (Cheewangkoon et al. 2010). The seven isolate were 95.8%, 99-100% and 99.8% identical to Pseudoplagiostoma mangiferae Dayarathne, Phookamsak & K. D. Hyde KUMCC 18-0179 (the ex-type strain) for the ITS gene (MK084824), TUB2 gene (MK084823) and LSU gene (MK084825), respectively. Phylogenetic analysis based on concatenated sequences of ITS and LSU genes was performed by the Maximum Likelihood method. All seven isolates were clustered in a well-supported clade with P. mangiferae KUMCC 18-0179 with 100% bootstrap value. Based on the pathogenicity and morphological characteristics, the pathogen was identified as P. mangiferae which was reported as a new species associated with mango leaf blight in Yunnan, China (Bezerra et al. 2019; Cheewangkoon et al. 2010; Crous et al. 2012; Crous et al. 2018; Phookamsak et al. 2019; Suwannarach et al. 2016). The newly emerging leaf blotch may become a prevalent disease of mango in future.

First report of Septoria blotch of passion fruit caused by Septoria passifloricola in Taiwan.

Passion fruit originated in South America and cultivated in tropical and subtropical countries for the fresh market and juice processing. In Taiwan, healthy grafted seedlings of passion fruit have been used for replanting every year to minimize the impact of viral and root diseases. The grafted seedlings commonly used purple passion fruit 'Tainung NO.1' (Passiflora edulis × Passiflora edulis forma flavicarpa) abbreviated as PPF as scion, and yellow passion fruit (P. edulis f. flavicarpa) abbreviated as YPF as rootstock. In July 2016 and May 2018, a new leaf disease of passion fruit was observed in Taichung City and Nantou County on 2 to 3-month-old grafted passion fruit seedlings. About 17% of seedlings showed symptoms on leaves in a commercial greenhouse nursery. The infected leaves abscised earlier, causing reduced survival of grafted seedlings. The leaf lesions on YPF and PPF were round to irregular and white-grayish or light brown, and were surrounded by dark green borders and obvious chlorotic halos. Fungal pycnidia were formed in the center of lesions, and extruded yellow-white long conidial tendrils under high humidity. The presumed fungal pathogens were obtained by single spore isolation. Six isolates from the two geographic regions with similar morphological characteristics on potato dextrose agar were obtained. To confirm the pathogenicity, YPF seedlings were inoculated by dropping 10 µL of a conidial suspension of isolate PLS-S2 (107 conidia/mL) on each inoculation site located on abaxial leaves surfaces that were either intact or wounded to form 3 pinpricks in a 4 mm area with a sterilized needle. Three plants were used in a treatment and four leaves of each plant were inoculated. The inoculated plants were kept in plastic bags with high humidity for 3 days and grown in a walk-in growth chamber at 24℃ with a 12-h light regime. The initial symptoms were punctate lesions that later enlarged to round, necrotic spots surrounded by yellow halos, which resembled symptoms in commercial greenhouse nurseries. About 44% of inoculation sites (n= 48) on intact leaves developed lesions at 28 days post-inoculation (dpi) while 100% of inoculation sites (n= 72) on wounded leaves showed lesions at 21 dpi. No lesions developed on leaves with water control. Pathogens reisolated from these lesions were morphologically identical to the inoculated fungus. Conidia were hyaline, filiform to cylindrical with 1-3 nonconstricted septa, and mostly 9-30 × 1.0-2.3 µm. The morphological characteristics of the isolates were similar to Septoria passifloricola Punith (Cline, 2006). Molecular identification was based on concatenated sequences of partial TEF1-α gene (accession nos. MK643056 to MK643061) and ß-tubulin gene (accession nos. MK643050 to MK643055) for each of the six isolates. The BLAST search revealed that strain PLS-S2 was 100.0% identical (392 bp) to S. passifloricola CBS 129431 for the TEF1-α gene (KF253443.1) and 98.4% identical (311 bp) for the ß-tubulin gene (KF252964.1). Phylogenetic analysis showed that PLS-S2 and five additional isolates clustered with reference strains of S. passifloricola (Verkley et al. 2013) in a well-supported clade (95% bootstrap value). Results suggested that the leaf disease of passion fruit in Taiwan was caused by S. passifloricola. This disease has been reported in Africa, India, Australia, New Zealand, Caribbean, and South America (Cline 2006; Ploetz et al. 2003). If appropriate control actions are not taken, the disease may become a major leaf disease in nurseries in Taiwan.

Artificial Intelligence for Automatic Measurement of Sagittal Vertical Axis Using ResUNet Framework.

We present an automated method for measuring the sagittal vertical axis (SVA) from lateral radiography of whole spine using a convolutional neural network for keypoint detection (ResUNet) with our improved localization method. The algorithm is robust to various clinical conditions, such as degenerative changes or deformities. The ResUNet was trained and evaluated on 990 standing lateral radiographs taken at Chang Gung Memorial Hospital, Linkou and performs SVA measurement with median absolute error of 1.183 ± 0.166 mm. The 5-mm detection rate of the C7 body and the sacrum are 91% and 87%, respectively. The SVA calculation takes approximately 0.2 s per image. The intra-class correlation coefficient of the SVA estimates between the algorithm and physicians of different years of experience ranges from 0.946 to 0.993, indicating an excellent consistency. The superior performance of the proposed method and its high consistency with physicians proved its usefulness for automatic measurement of SVA in clinical settings.

Identification and trichothecene genotypes of Fusarium graminearum species complex from wheat in Taiwan.

BACKGROUND: Fusarium head blight (FHB) of wheat caused by Fusarium graminearum species complex (FGSC) is a devastating disease worldwide. The pathogens not only reduce the yield of wheat, but also impact the quality of wheat by contamination with trichothecene mycotoxins. A systematic investigation on the pathogens of FHB in Taiwan is lacking. Here, molecular and morphological approaches were used to identify species of the Taiwanese FGSC isolates and determine their trichothecene genotypes. RESULTS: In this study, a total of 195 isolates of FGSC from diseased wheat were collected from 8 areas of northern and central Taiwan. All isolates were subjected to seedling inoculation for verification of pathogenicity. The pathogenic isolates were genetically characterized by sequence characterized amplified region (SCAR), PCR- restriction fragment length polymorphism (RFLP), phylogenetic analysis and fixed nucleotides to clarify their phylogenetic species, and by PCR assays of TRI genes to determine trichothecene genotypes. They were identified as F. asiaticum, F. graminearum sensu stricto, F. meridionale and an unknown species. Isolates of F. asiaticum were the major causal agents (98%) in this investigated population and were comprised of SCAR type 5 (75%), SCAR type 4 (21%) and SCAR type 3 (2%). Their trichothecene genotypes were either 15-acetyl-deoxynivalenol (15-ADON) (83%) or nivalenol (NIV) genotype (17%). These genetic characterizations indicated that F. asiaticum (15-ADON SCAR type 5) accounts for 60% of this Taiwanese population. Virulence assay on wheat heads indicated virulence of F. asiaticum isolates in subpopulations divided by SCAR types or trichothecene genotypes were comparable, suggesting other factors influence the unequal subpopulation sizes. CONCLUSIONS: This is the first study that FGSC isolates in Taiwan were systematically collected and characterized. In addition to F. graminearum sensu stricto and F. meridionale, F. asiaticum with 15-ADON genotype was identified as the predominate species in Taiwan. In contrast to Chinese and Japanese populations that F. asiaticum isolates were typically of 3-ADON or NIV genotype, the predominate 15-ADON genotype in Taiwanese population was unique among F. asiaticum populations and represented the southernmost 15-ADON genotype population in East Asia.

YAP1 homologue-mediated redox sensing is crucial for a successful infection by Monilinia fructicola.

Monilinia fructicola (G. Winter) Honey is a devastating pathogen on Rosaceae which causes blossom blight and fruit rot. Only a few studies related to the plant-pathogen interaction have been published and there is limited knowledge on the relationship between oxidative stress and successful infection in M. fructicola. In this study, we cloned and characterized a redox-responsive transcription factor MFAP1, a YAP1 homologue. MfAP1-silenced strains were generated by polyethylene glycol-mediated protoplast transformation or Agrobacterium T-DNA-mediated transformation. Pathogenicity assay demonstrated that MfAP1-silenced strains caused smaller lesions on rose and peach petals. Transformants carrying extra copies of MfAP1, driven by the native promoter, were generated for MfAP1 overexpression. Interestingly, MfAP1-overexpressing strains also caused smaller lesions on rose petals. Strains carrying two copies of MfAP1 accumulated reactive oxygen species (ROS) at higher levels and exhibited delayed accumulation of MfAP1 transcripts compared with the wild-type during pathogenesis. By the analysis of ROS production and the expression patterns of redox- and virulence-related genes in the wild-type strain and an MfAP1-overexpressing strain, we found that the M. fructicola wild-type strain responded to oxidative stress at the infection site, activated the expression of MfAP1 and up-regulated the genes required for ROS detoxification and fungal virulence. In contrast, MfAP1 expression in the MfAP1-overexpressing strain was suppressed after the induction of a strong oxidative burst at the infection site, altering the expression of ROS detoxification and virulence-related genes. Our results highlight the importance of MfAP1 and ROS accumulation in the successful infection of M. fructicola.

The Genomes of Three Uneven Siblings: Footprints of the Lifestyles of Three Trichoderma Species.

The genus Trichoderma contains fungi with high relevance for humans, with applications in enzyme production for plant cell wall degradation and use in biocontrol. Here, we provide a broad, comprehensive overview of the genomic content of these species for "hot topic" research aspects, including CAZymes, transport, transcription factors, and development, along with a detailed analysis and annotation of less-studied topics, such as signal transduction, genome integrity, chromatin, photobiology, or lipid, sulfur, and nitrogen metabolism in T. reesei, T. atroviride, and T. virens, and we open up new perspectives to those topics discussed previously. In total, we covered more than 2,000 of the predicted 9,000 to 11,000 genes of each Trichoderma species discussed, which is >20% of the respective gene content. Additionally, we considered available transcriptome data for the annotated genes. Highlights of our analyses include overall carbohydrate cleavage preferences due to the different genomic contents and regulation of the respective genes. We found light regulation of many sulfur metabolic genes. Additionally, a new Golgi 1,2-mannosidase likely involved in N-linked glycosylation was detected, as were indications for the ability of Trichoderma spp. to generate hybrid galactose-containing N-linked glycans. The genomic inventory of effector proteins revealed numerous compounds unique to Trichoderma, and these warrant further investigation. We found interesting expansions in the Trichoderma genus in several signaling pathways, such as G-protein-coupled receptors, RAS GTPases, and casein kinases. A particularly interesting feature absolutely unique to T. atroviride is the duplication of the alternative sulfur amino acid synthesis pathway.

The Colletotrichum graminicola striatin orthologue Str1 is necessary for anastomosis and is a virulence factor.

Striatin family proteins are key regulators in signalling pathways in fungi and animals. These scaffold proteins contain four conserved domains: a caveolin-binding domain, a coiled-coil motif and a calmodulin-binding domain at the N-terminus, and a WD-repeat domain at the C-terminus. Fungal striatin orthologues are associated with sexual development, hyphal growth and plant pathogenesis. In Fusarium verticillioides, the striatin orthologue Fsr1 promotes virulence in the maize stalk. The relationship between fungal striatins and pathogenicity remains largely unexplored. In this study, we demonstrate that the Colletotrichum graminicola striatin orthologue Str1 is required for full stalk rot and leaf blight virulence in maize. Pathogenicity assays show that the striatin mutant strain (Δstr1) produces functional appressoria, but infection and colonization are attenuated. Additional phenotypes of the Δstr1 mutant include reduced radial growth and compromised hyphal fusion. In comparison with the wild-type, Δstr1 also shows a defect in sexual development and produces fewer and shorter conidia. Together with the fact that F. verticillioides fsr1 can complement Δstr1, our results indicate that C. graminicola Str1 shares five phenotypes with striatin orthologues in other fungal species: hyphal growth, hyphal fusion, conidiation, sexual development and virulence. We propose that fungal striatins, like mammalian striatins, act as scaffolding molecules that cross-link multiple signal transduction pathways.

F-actin localization dynamics during appressorium formation in Colletotrichum graminicola.

Appressoria are essential penetration structures for many phytopathogenic fungi. Here F-actin localization dynamics were documented during appressorium formation in vitro and in planta in Colletotrichum graminicola Four discernible stages of dynamic F-actin distribution occurring in a programmed order were documented from differentiation of appressoria to formation of penetration pores: (stage A) from germ tube enlargement to complete expansion of the appressorium; (stage S) septation occurs; (stage L) a long period of low F-actin activity; (stage P) the penetration pore forms. The F-actin subcellular localization corresponded to each stage. A distinct redistribution of actin cables occurred at the transition from stage A to stage S. The in planta assays revealed that F-actin also assembled in invasive hyphae and that actin cables might play an essential role for penetration-peg development. The F-actin localization distribution may be used as a subcellular marker to define the developmental stages during appressorium formation.

Blue light acts as a double-edged sword in regulating sexual development of Hypocrea jecorina (Trichoderma reesei).

The industrially important cellulolytic filamentous fungus Trichoderma reesei is the anamorph of the pantropical ascomycete Hypocrea jecorina. H. jecorina CBS999.97 strain undergoes a heterothallic reproductive cycle, and the mating yields fertilized perithecia imbedded in stromata. Asci in the perithecia contain 16 linearly arranged ascospores. Here, we investigated H. jecorina sexual development under different light regimes, and found that visible light was dispensable for sexual development (stroma formation and ascospore discharge). By contrast, constant illumination inhibited stroma formation, and an interruption of the darkness facilitated timely stroma formation in a 12 h/12 h light-dark photoperiod. The results of genetic analyses further revealed that H. jecorina blue-light photoreceptors (BLR1, BLR2) and the photoadaptation protein ENV1 were not essential for sexual development in general. BLR1, BLR2 and ENV1 are orthologues of the conserved Neurospora crassa WC-1, WC-2 and VVD, respectively. Moreover, BLR1 and BLR2 mediate both positive and negative light-dependent regulation on sexual development, whereas ENV1 is required for dampening the light-dependent inhibitory effect in response to changes in illumination. Comparative genome-wide microarray analysis demonstrated an overview of light-dependent gene expression versus sexual potency in CBS999.97 (MAT1-2) haploid cells. Constant illumination promotes abundant asexual conidiation and high levels of hpp1 transcripts. hpp1 encodes a h (hybrid)-type propheromone that exhibits features of both yeast a and a pheromone precursors. Deletion of hpp1 could rescue stroma formation but not ascospore generation under constant illumination. We inferred that the HPP1-dependent pheromone signaling system might directly prevent stroma formation or simply disallow the haploid cells to acquire sexual potency due to abundant asexual conidiation upon constant illumination.

A role for endocytic recycling in hyphal growth.

Actin plays multiple complex roles in cell growth and cell shape. Recently it was demonstrated that actin patches, which represent sites of endocytosis, are present in a sub-apical collar at growing tips of hyphae and germ tubes of filamentous fungi. It is now clear that this zone of endocytosis is necessary for filamentous growth to proceed. In this review evidence for the role of these endocytic sites in hyphal growth is examined. One possibility if that the role of the sub-apical collar is associated with endocytic recycling of polarized material at the hyphal tip. The 'Apical Recycling Model' accounts for this role and predicts the need for a balance between endocytosis and exocytosis at the hyphal tip to control growth and cell shape. Other cell differentiation events, including appressorium formation and Aspergillus conidiophore development may also be explained by this model.

Aspergillus nidulans striatin (StrA) mediates sexual development and localizes to the endoplasmic reticulum.

Striatin family proteins have been identified in animals and fungi and are considered to be scaffolding proteins. In fungi striatin orthologs have been associated with sexual development and virulence to plants. In this study, we characterized the functions and localization of the striatin ortholog, StrA, in Aspergillus nidulans. deltastrA strains showed multiple defects in conidium germination, mycelial radial growth, production of diffusible red pigment, and reduced conidiation. The most striking phenotype is the production of abnormally small cleistothecia that are defective in ascosporogenesis. Over-expression of strA enhanced cleistothecium development and increased the production of Hülle cells in shaking liquid cultures. In addition, we generated strains expressing StrA::eGFP under the endogenous promoter. By co-labeling with FM4-64 and co-localization with nuclear localized StuA(NLS)::DsRed or CxnA (an endoplasmic reticulum marker), we determined that StrA mainly localizes to endoplasmic reticulum and the nuclear envelope.

Diversity of the trifunctional histidine biosynthesis gene (his) in cereal Phaeosphaeria species.

Phaeosphaeria species are important causal agents of Stagonospora leaf blotch diseases in cereals. In this study, the nucleotide sequence and deduced polypeptide of the trifunctional histidine biosynthesis gene (his) are used to investigate the phylogenetic relationships and provide molecular identification among cereal Phaeosphaeria species. The full-length sequences of the his gene were obtained by PCR amplification and compared among cereal Phaeosphaeria species. The coding sequence of the his gene in wheat-biotype P. nodorum (PN-w) was 2697 bp. The his genes in barley-biotype P. nodorum (PN-b), two P. avenaria f. sp. triticea isolates (homothallic Pat1 and Pat3), and Phaeosphaeria species from Polish rye and dallis grass were 2694 bp. The his gene in heterothallic isolate Pat2, however, was 2693 bp because the intron had one fewer base. In P. avenaria f. sp. avenaria (Paa), the his gene was only 2670 bp long. The differences in the size of the his gene contributed to the variation in amino acid sequences in the gap region located between the phosphoribosyl-ATP pyrophosphohydrolase and histidinol dehydrogenase sub-domains. Based on nucleotide and deduced amino acid sequences of the his gene, Pat1 was not closely related to either PN-w or the Paa clade. It appears that rates of evolution of the his gene were fast in cereal Phaeosphaeria species. The possible involvement of meiotic recombination in genetic diversity of the his gene in P. nodorum is discussed.