First report of jujube anthracnose caused by Colletotrichum siamense in China.

Jujube (Zizyphus jujuba Mill.), a native small deciduous tree of China, is widely cultivated in China, Korea, India, Japan, Europe, and the United States (Chen et al. 2020). The fruit have been commonly consumed as healthy food supplements and traditional Chinese medicine for over 2000 years (Li et al. 2007). In August 2019, anthracnose-like leaf spot symptoms were observed on jujube plants in Xiaomenya Village, Jinan City, Shandong Province, China (36°27'39″N, 117°3'13″E), with over 30% leaf disease incidence. The spots were circular, sunken, brown in the center and with dark brown edges. As the spots enlarged and coalesced, it resulted in leaf perforation and early defoliation. Sometimes acervuli were observed on the lesions (Fig. S1a, b). To identify the causal agent, 20 diseased leaves were sampled, the margins of the lesions were cut into pieces (5 × 5 mm), sterilized and cultured following the protocol described previously (Wan et al. 2020) at 25 ℃ for 5 days. Twelve monospore isolates showing identical colony morphology were obtained. Three representative isolates, JNZG11, JNZG311, JNZG313, were used for further study. When grown on PDA the colony color was initially white and then turned pale-gray to gray in 5-day-old cultures. On the reverse, colonies were brown-black with an orange pigmentation near the center. Aerial mycelium was cottony, dense, white to pale-gray. Conidia were hyaline, 1-celled, smooth-walled, subcylindrical, oblong, attenuated with slightly rounded ends, (11.1-) 12.7-13.3 (-17.8) ×(-4.4) 5.2-5.5 (-6.3) µm (n=50). Appressoria were dark-brown, oval or irregular, (7.3-) 8.6-9.2 (-9.8) ×(-5.1) 5.8-6.9 (-7.0) µm (n=50) (Fig. S1c-g). The morphology resembled those of Colletotrichum gloeosporioides species complex (Cannon et al. 2012). For accurate identification, the sequences of the ribosomal internal transcribed spacer (ITS), actin (ACT), ß-tub2 (TUB2), calmodulin (CAL), chitin synthase (CHS-1), and glyceraldehyde-3phosphate dehydrogenase (GAPDH) of the 3 isolates were sequenced (Weir et al. 2012), and deposited into GenBank (Accession Nos. see Table 1). The six loci (ITS, GAPDH, ACT, CHS-1, CAL, and TUB2) were concatenated and the aligned sequences (1904 bp) were 99.7% homologous to ex-type C. siamense ICMP18578. The sequences of 38 Colletotrichum species (44 isolates) were downloaded from GenBank for phylogenetic analyses. In the maximum likelihood phylogenetic tree generated, the highest log likelihood was -8798.90 and the three isolates were all in the C. siamense clade (bootstrap support 94 %) (Fig. S2). To complete Koch's postulates, 60 healthy, mature jujube leaves on 12 branches (5 leaves per branch) (variety 'Zhongqiuhong') were inoculated with 20 µL of spore suspension (106 conidia/mL) or sterile water as a control. The branches were placed in sterile beakers containing a small amount of sterile water sealed with plastic wrap and maintained at 28 °C, 12 h light/dark. Five days after inoculation, all treated leaves showed the typical anthracnose symptom, similar to that observed in the field (Fig. S1h). The same fungus was re-isolated from the margins of the lesions using the aforementioned methods. Whereas no fungus were isolated from the controls. Previously, C. siamense has been reported to infect Z. mauritiana in China (Shu et al. 2020). To our knowledge, this is the first report of C. siamense causing anthracnose on Z. jujuba in China. This finding provides crucial information for the effective management of this disease.

First report of Colletotrichum siamense causing leaf spot on Michelia macclurei in China.

Michelia macclurei Dandy is an excellent timber and ornamental tree native to South China (Lan et al. 2010). In May 2020, a leaf spot disease of M. macclurei was found on the campus of Jiangxi Agricultural University (N28°45'56″, E115°50'21″). Approximately 25% (9 out of 35) of 32-year-old M. macclurei trees showed the leaf spot disease. On average, 40% of the leaves per individual tree were affected. The symptoms began as small dark brown lesions formed along the leaf margins and tips. The lesions' center was sunken with a dark brown border as the disease developed. Thirty pieces (5 × 5 mm) from the lesion margins were surface sterilized in 70% ethanol (30 s), then in 3% NaOCl (1 min), and finally rinsed three times with sterile water. Leaf pieces were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation. Sixteen strains with similar morphological characterizations were isolated, and three representative isolates (HX-1, HX-2, HX-3) were used for morphological and molecular characterization. The three isolates were white, cottony, and light gray on the reverse, producing dark-green pigmentation near the center. The conidia were single-celled, straight, hyaline, cylindrical, clavate, and measured 12.8-17.5 × 4.5-5.7 µm (14.7 ± 1.2 × 4.8 ± 0.2 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, and ranged from 5.9-8.8 × 4.4-6.7 µm (7.1 ± 0.6 × 5.6 ± 0.6 µm, n=100). The internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin2 (TUB2) were sequenced using the primers ITS1/ITS4 (White et al. 1990), ACT-512F/ACT-783R, CL1/CL2, CHS-79F/CHS-345R, GDF/GDR, and T1/Bt2b, respectively (Weir et al. 2012). The sequences were deposited into GenBank (Accession Nos.: MZ323328, MZ323329, MW581269 for ITS, MZ889002, MZ889003, MW661166 for ACT, MZ889004, MZ889005, MW661167 for CAL, MZ889006, MZ889007, MW661168 for CHS-1, MZ889008, MZ889009, MW661169 for GAPDH, MZ889010, MZ889011, MW661170 for TUB2). A maximum likelihood and Bayesian posterior probability-based analyses using IQ-tree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences (ITS, ACT, CAL, CHS-1, GAPDH, and TUB2 ) placed three isolates in the clade of Colletotrichum siamense Prihastuti, L. Cai & K. D. Hyde. Based on the morphological characteristics and molecular data, three isolates were identified as C. siamense (Fu et al. 2019).The pathogenicity of each isolate was tested on potted 2-yr-old seedlings of M. macclurei grown in a greenhouse at 25 ℃, 70% relative humidity with a 12-h photoperiod. Twenty healthy leaves on 10 M. macclurei plants were inoculated with 10 µL of spore suspension (106 conidia/mL). Another 20 healthy leaves were inoculated with sterile water as the control. All leaves were wounded with a sterile needle (Φ=0.5 mm). The resulting symptoms were similar to those on the original infected plants, whereas the control leaves remained asymptomatic for 8 days after inoculation. C. siamense was consistently re-isolated only from the diseased leaves, fulfilling Koch's postulates. C. siamense can cause leaf diseases in a variety of hosts, such as Salix matsudana (Zhang et al. 2021), Liriodendron chinense [Hemsl.] Sarg. × tulipifera L. (Zhu et al. 2019) and Magnolia grandiflora (Zhou et al. 2022). This is the first report of C. siamense associated with leaf spot disease on M. macclurei in China, and its potential threat should be evaluated in the future. These results will help to develop effective strategies for appropriately managing this newly emerging disease.

First Report of Leaf Spot Caused by Colletotrichum siamense on Magnolia grandiflora in China.

Magnolia grandiflora (Southern magnolia) is a popular evergreen tree, planted especially as an ornamental for landscaping. In September 2019, leaf spots were observed on M. grandiflora at the campus of Jiangxi Agricultural University (28°45'56″N, 115°50'21″E). Approximately 64% (23 out of 36) M. grandiflora trees (most 24-year-old) occurred leaf spot disease at the campus. On average, 40% of the leaves per individual tree were affected. Foliar symptoms began as small dark brown lesions formed along the leaf margins. As the disease developed, the lesions' center was sunken with a dark brown border. Symptomatic leaves were collected and cut into 5 × 5 mm pieces. Leaf pieces from the margin of the necrotic tissue were surface sterilized in 70% ethanol for 30 s followed by 2% NaOCl for 1 min and then rinsed in sterile water three times. Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Of more than 35 isolates, most shared a similar morphology, with an isolation rate of 85%. Three isolates (JNG-1, JNG-2, and JNG-3) were chosen for single-spore purification and used for morphological characterization and identification. Colonies on PDA of the three isolates were white, cottony, and grayish-white on the undersides of the culture. Conidia were single-celled, straight, hyaline, cylindrical, clavate, and measured 4.4-5.6 × 13.2-17.8 µm (4.7 ± 0.3 × 14.6 ± 1.0 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and ranged from 5.5-9.2 × 4.6-6.5 µm (7.3 ± 0.4 × 5.4 ± 0.3 µm, n=100). Morphological features were similar to Colletotrichum siamense as previously described (Weir et al. 2012). The internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), beta-tubulin 2 (TUB2), chitin synthase (CHS-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, ACT-512F/ACT-783R, CL1/CL2, T1/Bt2b, CHS-79F/CHS-345R and GDF/GDR (Weir et al. 2012), respectively and sequenced. All sequences were deposited into GenBank (ITS, MZ325948-MZ325950; ACT, MZ461477 - MZ461479; GAPDH, MZ461483 - MZ461485; TUB2, MZ461486 - MZ461488; CHS-1, MZ441182 - MZ441184; CAL, MZ461480 - MZ461482). A neighbor-joining phylogenetic tree was constructed with MEGA 7.0 using the concatenation of multiple sequences (Kumar et al. 2016). According to the phylogenetic tree, all three isolates fall within the C. siamense clade (boot support 96%). The pathogenicity of three isolates were tested on M. grandiflora plants, which were grown in the field. Healthy leaves were wounded with a sterile needle and then inoculated with 10 µL of spore suspension (106 conidia/mL). Controls were treated with ddH2O (Zhu et al. 2019). All the inoculated leaves were covered with black plastic bags to keep a high-humidity environment for 2 days. All the inoculated leaves showed similar symptoms to those observed in field, whereas control leaves were asymptomatic for 10 days. The infection rate was 100%. C. siamense was re-isolated from the lesions, whereas no fungus was isolated from control leaves. It was confirmed that C. gloeosporioides is the causal agent of leaf spot on Magnolia virginiana in America (Xiao et al. 2004). However, this is the first report of C. siamense causing leaf spot on M. grandiflora in China. This study provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

First report of leaf spot disease caused by Colletotrichum siamense on Cornus hongkongensis (Hemsl.) in China.

Cornus hongkongensis (Hemsl.) is an excellent ornamental tree species in China and elsewhere. In 2019, C. hongkongensis anthracnose was firstly observed at the campus of Jiangxi Agricultural University (JXAU) (28°45'56″N, 115°50'21″E), then found in parks, Nanchang, China. In early August, the disease appeared and lasted until the leaves dropped (November). The disease incidence was above 60%, and the diseased leaf rate was above 70%. The lesions mostly appeared along the leaf edges. Some small round to irregular lesions also developed in other parts of the leaves. These diseased leaves had circular or irregularly shaped spots with gray-white color in the center and dark brown on the edge of the lesions. Later, the lesions became necrotic and shriveled. As the disease progressed, the spots coalesced so that affected leaves appeared blighted (Supplementary Figure 1 A-C). To identify the pathogen, leaves with typical symptoms from the campus of JXAU were collected and small pieces (5 × 5 mm) from the lesion borders were surfaced sterilized in 70% ethanol for 30 s, followed by 1 min in 3% NaOCl, and then rinsed with sterile distilled water three times. Leaf pieces were placed on potato dextrose agar (PDA) and incubated at 25 °C under a 12-h light/dark cycle (3000 lx). Pure cultures were obtained from individual conidia by single spore isolates. For studies of microscopic morphology, a representative isolate JX-S4 was subcultured on PDA. The colony of JX-S4 was white and turning gray and light gray on the reverse side, producing dark-green pigmentation near the center (Supplementary Figure 1 D). The conidia were one-celled, straight, hyaline, subcylindrical with rounded ends and 16.9 ± 1.6 × 6.0 ± 0.6 µm (n = 50) in size. Appressoria were one-celled, pale brown, thick-walled, ellipsoidal, and measured 8.7 ± 1.7 × 6.4 ± 0.8 µm (n = 50) (Supplementary Figure 1 E, F). The morphological characteristics of JX-S4 matched those of the Colletotrichum siamense species (Weir et al. 2012). For accurate identification, the internal transcribed spacer (ITS) and the genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-I), beta-tubulin 2 (TUB2), and calmodulin (CAL) were respectively amplified with primers ITS1/ITS4, GDF/GDR, CHS-79F/CHS-345R, ßt2a/ßt2b, and CL1/CL2. The sequences were deposited in GenBank (Accession nos. MT587807, MT628710, MT628709, MT628711, and MT628708). Phylogenetic analysis was calculated with concatenated sequences (ITS, GAPDH, CHS-I, CAL, and TUB2) using MEGA 7. In the maximum likelihood phylogenetic tree, Isolate JX-S4 was clustered with C. siamense with 93% bootstrap support (Supplementary Figure 2). Based on the morphological characteristics and phylogenetic analysis, JX-S4 was identified as C. siamense. Pathogenicity test of JX-S4 was verified on 45 attached healthy leaves from three C. hongkongensis plants (10-year-old) at the campus of JXAU inoculated with mycelial plugs (φ=5 mm) from the culture edge (6-day-old) on PDA. And an additional 45 healthy leaves were inoculated with PDA plugs as controls. The leaves were wounded with a red-hot needle (φ=0.5 mm). All treatment and control leaves were wrapped up with black plastic bags to keep them moist for 2 days. The pathogenicity tests were repeated twice. Within 7 days, all the inoculated leaves developed the lesions, which were similar to those observed in the field. Control leaves were asymptomatic (Supplementary Figure 1 G, H). The same fungus was re-isolated from the symptomatic tissues, fulfilling Koch's postulates. To our knowledge, this is the first report of C. siamense causing C. hongkongensis anthracnose. This finding provides crucial information for managing this disease. For example, when diagnosing Cornus anthracnose, C. siamense needs to be looked out for and appropriate control measures implemented.

Identification, Virulence and Fungicide Sensitivity of Colletotrichum gloeosporioides s.s. Responsible for Walnut Anthracnose Disease in China.

Walnut (Juglans regia L.) is an economically important woody nut and edible oil tree all over the world. However, walnut production is limited by walnut anthracnose, which is a disastrous disease that causes significant yield losses. Studying the etiology of anthracnose on walnut and the pathogens' virulence and sensitivities to fungicides would be beneficial for effective control. This study was conducted to identify the pathogen of walnut anthracnose and reveal the population diversity of pathogens through virulence, sensitivities to fungicides, and genetic variation. A total of 13 single-spore Colletotrichum isolates were collected from walnut anthracnose-diseased fruits and leaves from 13 walnut commercial orchards in Henan, Hubei, Shandong, and Shaanxi provinces in China. The isolates were identified as Colletotrichum gloeosporioides sensu stricto (s.s.) according to multilocus phylogenetic analyses (internal transcribed spacer, actin, glyceraldehyde-3-phosphate dehydrogenase, and chitin synthase), morphological as well as cultural characters, and pathogenicity. When the same walnut tissue was inoculated with different isolates, the disease lesion size was different. The results showed that the virulence of all isolates was considerably different, and the differences were not correlated with geographic origins. The virulence to walnut leaves and fruits inoculated with the same isolate was significantly different. Based on the virulence to walnut leaves and fruits, the 13 isolates were divided into three groups. Virulence of 69.2% of the isolates to walnut fruits was higher than that to leaves; 15.4% of isolates had no difference in pathogenicity, and the virulence to walnut leaves was higher for 15.4% of isolates. Tebuconazole, difenoconazole, flusilazole, and carbendazim inhibited the growth of fungal mycelia, and the concentration for 50% of maximal effect (EC50) values were 0.4 to 20.5, 0.6 to 2.6, 0.2 to 1.6, and 0.002 to 0.2 µg/ml, respectively, with average values of 6.5 ± 6.9, 1.5 ± 0.6, 0.9 ± 0.4, and 0.1 ± 0.05 µg/ml, respectively. All isolates were more sensitive to difenoconazole, flusilazole, and carbendazim than tebuconazole (P < 0.01). Isolate sensitivities to the same fungicide were different. Isolates SL-31 and TS-09 were the least sensitive to carbendazim and tebuconazole, respectively, and the resistance ratios were 87.3 and 51.6, respectively. Sensitivities to difenoconazole and flusilazole were largely consistent among all isolates, and the resistance ratios were from 1 to 4.6 and from 1 to 7, respectively. Therefore, difenoconazole and flusilazole could be chosen for disease control. The differences of pathogenicity and fungicide sensitivity were not correlated with geographic regions. These results indicated that there was high intraspecific diversity of populations in C. gloeosporioides s.s. that caused walnut anthracnose. For effective management, the targeted control strategy should be implemented based on the different geographic regions.

Long-Term Carriage of Medicopsis romeroi, an Agent of Black-Grain Mycetoma, Presenting as Phaeohyphomycosis in a Renal Transplant Patient.

Medicopsis species are rare fungal pathogens that frequently resist common antifungal therapies and are difficult to identify morphologically as conidia are produced in pycnidia, a key feature of coelomycetes. Immunocompromised patients are at risk of these infections, even after remote exposure, and typically present with phaeohyphomycoses without dissemination. We present the case of a renal transplant recipient 6.5 years post-transplant who developed a slowly progressive soft tissue infection mimicking a synovial cyst. A cultured isolate was identified as Medicopsis romeroi by sequencing of multiple ribosomal loci. The patient responded well to debridement and posaconazole therapy. Solid-organ transplant patients are at risk of opportunistic fungal infection long after transplant, and molecular methods are often required for definitive identification.

In vitro antifungal susceptibility of coelomycete agents of black grain eumycetoma to eight antifungals.

Fungal mycetoma (eumycetoma) represents one of the most difficult infections to appropriately manage. The current recommended treatment is based on extensive surgical debridement combined with prolonged antifungal therapy with ketoconazole or itraconazole. Despite the different phylogenetic positions of black-grain eumycetoma species, they are all treated with the same antifungal agents. The in vitro antifungal susceptibility of coelomycetous eumycetoma agents in the order of Pleosporales presently is largely unknown. Here we determined the in vitro activity of eight antifungal agents against seven species causing human eumycetoma using the Sensititre YeastOne method. High minimum inhibitory concentrations (MICs) were found with fluconazole, caspofungin, flucytosine, and amphotericin B. Voriconazole and posaconazole were found to be active against all species tested. Of the species included in the investigation, MICs of Medicopsis romeroi differed from the rest of the mycetoma causative agents belonging to the order of the Pleosporales. We found significantly lower MICs for amphotericin B and significantly higher MICs for fluconazole, ketoconazole, and itraconazole against this species. Our results emphasised that identification of black grain mycetoma agent is important as well as performing susceptibility testing before starting of antifungal treatment.

Phacidium and Ceuthospora (Phacidiaceae) are congeneric: taxonomic and nomenclatural implications.

The morphologically diverse genus Ceuthospora has traditionally been linked to Phacidium sexual morphs via association, though molecular or cultural data to confirm this relationship have been lacking. The aim of this study was thus to resolve the relationship of these two genera by generating nucleotide sequence data for three loci, ITS, LSU and RPB2. Based on these results, Ceuthospora is reduced to synonymy under the older generic name Phacidium. Phacidiaceae (currently Helotiales) is suggested to constitute a separate order, Phacidiales (Leotiomycetes), as sister to Helotiales, which is clearly paraphyletic. Phacidiaceae includes Bulgaria, and consequently the family Bulgariaceae becomes a synonym of Phacidiaceae. Several new combinations are introduced in Phacidium, along with two new species, P. pseudophacidioides, which occurs on Ilex and Chamaespartium in Europe, and Phacidium trichophori, which occurs on Trichophorum cespitosum subsp. germanicum in The Netherlands. The generic name Allantophomopsiella is introduced to accommodate A. pseudotsugae, a pathogen of conifers, while Gremmenia is resurrected to accommodate the snow-blight pathogens of conifers, G. abietis, G. infestans, and G. pini-cembrae.

Septoria-like pathogens causing leaf and fruit spot of pistachio.

Several species of Septoria are associated with leaf and fruit spot of pistachio (Pistacia vera), though their identity has always been confused, making identification problematic. The present study elucidates the taxonomy of the Septoria spp. associated with pistachio, and distinguishes four species associated with this host genus. Partial nucleotide sequence data for five gene loci, ITS, LSU, EF-1α, RPB2 and Btub were generated for a subset of isolates. Cylindroseptoria pistaciae, which is associated with leaf spots of Pistacia lentiscus in Spain, is characterised by pycnidial conidiomata that give rise to cylindrical, aseptate conidia. Two species of Septoria s. str. are also recognised on pistachio, S. pistaciarum, and S. pistaciae. The latter is part of the S. protearum species complex, and appears to be a wide host range pathogen occurring on hosts in several different plant families. Septoria pistacina, a major pathogen of pistachio in Turkey, is shown to belong to Pseudocercospora, and not Septoria as earlier suspected. Other than for its pycnidial conidiomata, it is a typical species of Pseudocercospora based on its smooth, pigmented conidiogenous cells and septate conidia. This phenomenon has also been observed in Pallidocercospora, and seriously questions the value of conidiomatal structure at generic level, which has traditionally been used to separate hyphomycetous from coelomycetous ascomycetes. Other than DNA barcodes to facilitate the molecular identification of these taxa occurring on pistachio, a key is also provided to distinguish species based on morphology.