Soil ascomycetes from Spain. XIV. The Chaetomiaceae of La Palma (Canary Islands).

We analyzed the sequences of three DNA regions—the translation elongation factor-1 alpha (EF-1 alpha) gene and the internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of ribosomal DNA—to compare their accuracy in identifying species of Japanese Armillaria. We studied 49 isolates of eight Armillaria species, A. mellea, A. ostoyae, A. nabsnona, A. cepistipes, A. gallica, A. sinapina, A. tabescens and the biological species Nagasawa E (Nag. E). Phylogenetic analyses of the ITS and IGS data helped in identifying A. mellea, A. ostoyae, A. nabsnona, A. tabescens and Nag. E but could not be used to identify A. gallica, A. cepistipes and A. sinapina. Nevertheless our analysis showed that the EF-1 alpha gene was clearly different in the eight examined species. Restriction fragment length polymorphisms (RFLP) of the IGS-1 region could be used to distinguish most species, but the RFLP profiles of some isolates of A. cepistipes and A. sinapina were the same even with four different restriction enzymes. In conclusion, among the techniques examined in this study, analyzing the EF-1 alpha sequence was found to be the most suitable method for identifying different species of Japanese Armillaria.

Pear (Pyrus communis) is an important fruit crop in the Netherlands, with a total production of 400,000 tons in 2020, and 'Conference' is the main pear cultivar that comprises 80% of total pear production area. In the Netherlands, pears are kept in controlled atmosphere cold storage (-0.5°C) up to 11 months after harvest. Calyx-end rot incidences of 1% to 5% were observed on 'Conference' pears from different orchards in surveys from 2019-2021 in packing houses in the Netherlands. Infections showed 1 to 3 cm brown necrosis. Lesions were round, slightly sunken and next to or including part of the calyx. To isolate the causal agent, fruit were rinsed with sterile water, lesions were sprayed with 70% ethanol until droplet runoff, the skin was removed aseptically with a scalpel, and tissue under the lesion was isolated and placed onto Potato Dextrose Agar (PDA) (Oxoid, UK). The PDA plates were incubated at 20°C in the dark, and hyphal tip isolates were transferred to fresh PDA plates. Colonies on PDA were rosy-whitish to peach-colored. Colonies grown on oat meal agar (OA) under UV light were peach to red color, aerial mycelium sparse, and produced a pink to salmon colored conidial matrix. Conidia were irregular-ellipsoidal to allantoid, smooth, hyaline and usually with one or several gutulles. Conidia were sometimes one septate and measured 15.2±2.8 x 4.0±0.7 μm (n =14), but mostly aseptate and measured 7.9±1.7 x 3.2±0.6 μm (n =100). The fungus was morphologically identical to Didymella macrostoma (syn. Phoma macrostoma) (Boerema et al. 2004; Hou et al. 2020). The identity of four representative isolates, WURR-206, WURR-223, WURR-227 and WURR-308, from affected pears from four orchards in the Netherlands, was determined by multilocus gene sequencing. To this end, genomic DNA was extracted using the LGC Mag Plant Kit (Berlin, Germany) in combination with the Kingfisher method (Waltham, MA). Sequences of the internal transcribed spacer (ITS) region of ribosomal DNA, the large-subunit rRNA (LSU) region, partial sequences of beta-tubulin (TUB) and the translation elongation factor 1-alpha (TEF1) gene region were amplified with primers ITS1/ITS4 (White et al. 1990), LROR/LR5 (Vilgalys and Hester 1990), Btub2Fd/Btub4Rd (Woudenberg et al. 2009) and EF1-983F/EF1-1567R (Rehner and Buckley 2005), respectively. Sequences were deposited under GenBank accession numbers ON077588-ON077591 (ITS), ON113487-ON113490 (LSU), ON098515-ON098518 (TUB) and ON098519-ON098522 (TEF1). MegaBLAST analysis revealed that the ITS, LSU, TUB sequences matched with 100% identity to culture collection sequences of Didymella macrostoma in GenBank MH854841 (ITS), MH866341 (LSU), MN983895 (TUB). The TEF1 sequences matched with 99.7% identity to TEF sequence of Didymella macrostoma MT454020. Subsequently, Koch's postulates were performed on 10 'Conference' pears per isolate (WURR-206, WURR-223, WURR-227 and WURR-308). Fruits wiped with 70% ethanol were inoculated in pathogenicity tests with an agar disk (5 mm diameter) of D. macrostoma prepared from the actively growing edge of 14-day-old cultures grown on PDA. Inoculated fruits were sealed in plastic bags and were incubated in darkness at 20°C. Typical symptoms appeared 7-10 days after inoculation on all pears. PDA-only controls remained symptomless. Fungal colonies isolated from the lesions and cultured on PDA morphologically resembled the original isolate from the infected pears. The identity of the re-isolations was confirmed as D. macrostoma by sequencing, thus completing Koch's postulates. To the best of our knowledge, this is the first report of D. macrostoma causing calyx-end rot of pears. The identification of this causal agent is important knowledge necessary for developing control measures for postharvest diseases of pear.

California is a major almond (Prunus dulcis) producer in the world. In September 2012, 2-year-old almond trees from an orchard in Fresno Co. with stem cankers were submitted for disease diagnosis. In a survey of the orchard, 12 ha (1,500 Nonpareil and 1,800 Monterey almond trees) of 48 ha trees had been killed apparently due to a stem canker. The cankers developed above the graft union, were covered with amber sap, and often girdled the trunk. Isolations made from tissues at the canker margins onto acidified potato dextrose agar (PDA) yielded two fungi, Macrophomina phaseolina (Tassi) Goid and Lasiodiplodia theobromae (Pat.) Griffon & Maubl (1). M. phaseolina and L. theobromae were isolated from eight and two of 10 cankered trees, respectively. No mixed infections were found. M. phaseolina isolates were characterized by gray hyphae that turned black with developing microsclerotia. L. theobromae isolates were characterized by white, aerial mycelium that turned mouse gray after 5 days. Young conidia were ellipsoidal, thick walled, initially hyaline, granular, and nonseptate; aged conidia were brown, 1-septate with longitudinal striations in the wall. Identity was confirmed by analyses of the internal transcribed spacer (ITS), β-tubulin 2 (BT2), and the translation elongation factor 1-alpha (TEF-1α) gene regions. BLAST searches at GenBank showed a high identity with reference sequences of type specimens both for M. phaseolina (isolates 7E64 to 7E69: ITS, 100%; BT2, 99%; TEF-1α, 99%) and L. theobromae (isolates 7E86 to 7E88: ITS, 99%; BT2, 99%; TEF-1α, 100%). Sequences of three gene regions were deposited as GenBank accessions KC357271 to KC357279 (ITS); KC357280 to KC357288 (BT2); and KC357289 to KC357297 (TEF-1α). The pathogenicity of M. phaseolina and L. theobromae to P. dulcis cultivars Butte, Carmel, Nonpareil, and Padre was investigated in an orchard at KARE using four isolates of M. phaseolina (7E64, 7E65, 7E66, and 7E69) and two isolates of L. theobromae (7E86 and 7E88). Ten 2-year-old branches per isolate from 7-year-old trees were inoculated with each isolate in late September 2012, after removing the bark with a 7-mm cork borer and placing a 7-day-old 7-mm-diameter agar plug bearing mycelium of each isolate directly into the fresh wound, mycelium side down. Ten additional branches of each of the four cultivars were inoculated with sterile PDA plugs and served as negative controls. Three weeks after inoculation, the average lesion produced by M. phaseolina on Butte, Carmel, Nonpareil, and Padre was 53, 52, 41, and 37 mm in length, respectively. Lesions produced by L. theobromae were 191, 206, 194, and 103 mm in length on the four cultivars, respectively. No disease lesion, only wounds, were produced on negative controls. Lesions produced by both pathogens were longer (P < 0.05) than wounds on the controls (average length 10 mm on all cultivars). Both L. theobromae isolates killed branches of cultivars Butte, Carmel, and Nonpareil in 2 weeks. M. phaseolina and L. theobromae were reisolated from the inoculated branches, and no fungus was reisolated from controls. Based on pathogenicity results, L. theobromae is more virulent to almond branches than M. phaseolina. To our knowledge, this is the second report of M. phaseolina (2) and the first report of L. theobromae as pathogens of P. dulcis trees in California.

Bletilla striata (named "Bai Ji" in Chinese) is a plant from the Orchidaceae family that has been employed in traditional Chinese medicine for thousands of years in China. Polysaccharides extracted from B. striata have been shown to have an effect on Alzheimer's disease (Lin et al. 2021). Since 2021, leaf spots have been observed in the B. striata plantation in Chongqing, China. Out of 200 plants, the disease incidence was estimated at 56%, and the disease index was estimated at 32%. The symptoms were necrotic lesions with brown edges and yellow halos; severe infection caused the infected leaves to become blighted, dry and fall off. To identify the causal agent, eighteen leaves with typical symptoms were collected from the B. striata plantation (30.60°N, 108.64°E). The margins of infected tissue areas were cut into small pieces (5×5 mm), surface sterilized with 70% ethanol for 1 min, and rinsed twice with sterile distilled water. The tissue was then surface sterilized in 3% sodium hypochlorite for 2 min, followed by three rinses with sterile water. The tissue was then placed onto potato dextrose agar (PDA) plates and incubated at 25°C for 3 days, pure cultures of fungal isolates were obtained by single-spore isolation, stored on PDA slants and maintained at 4°C. Colonies of the fungal isolates showed three color types, ranging from grayish white to green above with olive green on the reverse, but conidial characteristics were more similar and indicated this was a single fungus. Conidiophores were single, lateral from hyphae or terminal; straight or curved; smooth-walled with 1 to 8 septa; pale brown; usually with only one pigmented terminal conidiogenous site, sometimes with one additional lateral conidiogenous locus; sometimes slightly swollen at the apex; and 15 to 170 μm long, 2.5 to 4.5 μm wide. Conidia were in short or moderately long chains of 2-8 conidia normally, sometimes with more; rarely branched; normally 14.07 to 50 × 5.24 to 10 μm in size; ellipsoid, fusiform, long ellipsoid, obclavate or ovoid with 1 to 11 transverse septa and 2 to 4 longitudinal septa; beakless or with subcylindric or cylindric secondary conidiophores, analogous to the beak 4.25 to 58.6 μm long, 3.2 to 4.8 μm wide. The fungal isolates were tentatively identified as Alternaria sp. The representative isolate BJ8 was selected for the pathogenicity test. The leaves of six healthy plants of B. striata (two years old) grown in pots were washed with sterile water. Ten mL of conidial suspension (1×106 conidia mL-1) contained in 0.05% Tween 80 buffer was brushed onto upper and lower surfaces of all the leaves on three plants, while other plants were brushed with 10 mL 0.05% Tween 80 buffer to serve as controls. Plants were placed in a greenhouse at 25°C and 95±1% relative humidity after inoculation and observed for symptoms. The symptoms initially developed as irregular brown necrotic lesions on the inoculated leaves after 7 days, with a yellow halo around the lesions, consistent with the symptoms in the field. Leaves on the control plants did not produce any symptoms. For molecular identification, the genomic DNAs of representative isolates BJ5, BJ6, and BJ8 were extracted. The internal transcribed spacer (ITS) region and RNA polymerase II second largest subunit (RPB2), translation elongation factor 1-alpha (TEF1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were used for polymerase chain reaction (PCR), using primers ITS5/ITS4, GPD1/GPD2, EF-1F/EF-1B and RPB27cR/RPB25F2, respectively (White et al. 1990; Berbee and Pirseyedi et al. 1999; Carbone and Kohn 1999; Liu et al. 1999). The neighbor-joining tree revealed that these isolates are clustered together with the reference strain of A. burnsii. The sequences were deposited in NCBI GenBank BJ5 [ITS: OP897263; GAPDH: OQ544937; TEF1: OQ544941; RPB2: OQ544939], BJ6 [ITS: OP897262; GAPDH: OQ544938; TEF1: OQ544942; RPB2: OQ544940], and BJ8 [ITS: OK285209; GAPDH: OK340046; TEF1: OK340047; RPB2: OQ544936]. All three isolates showed 100% similarity with A. burnsii CBS 107.38 [ITS: KP124420; GAPDH: JQ646305; TEF1: KP125198; RPB2: JQ646457] ex-type sequence, thus the pathogen causing the leaf spot on B. striata was identified as A. burnsii. A. burnsii is an important pathogenic fungus causing blight of cumin (Shekhawat et al. 2013). Furthermore, Al-Nadabi et al. (2018) found that A. burnsii can cause leaf spots on wheat and date palms, and Sunapao et al. (2022) reported that A. burnsii can infect coconuts (Cocos nucifera), causing dirty panicle disease. This is the first report of A. burnsii causing leaf spot on B. striata in China. The new discovery shows that since A. burnsii can readily adapt to a variety of climatic conditions, controlling the fungus is crucial for the healthy growth of B. striata in the future. This study will provide a basis for further elucidating the pathogenic mechanism and development of effective control measures for this disease.

Species of Rudbeckia, often known as black-eyed Susan or coneflower, are used as bedding plants in UK gardens. The large, daisy-like flowerheads typically have bright yellow petals and a conspicuous black centre which may be raised into a cone shape. During the summer of 2016, Rudbeckia fulgida var. sullivantii cv. Goldsturm plants, purchased in the preceding May from a nursery in south-east England, were observed with black leaf spots in outdoor beds at the Royal Horticultural Society (RHS) Garden Wisley. Lower leaves exhibited small black lesions in July, which coalesced as the season progressed leading to complete necrosis of the bottom leaves and spotting on higher leaves. Spots showed no halos and became necrotic only later in the season. Flowering did not appear to be reduced. Pycnidia within the leaf spots were epiphyllous, 50-75 μm diameter, with a neck protruding slightly above the leaf surface. Conidia were filiform, 30-60 times 1.5-2 μm, with three septa. A single-spore isolate was obtained on water agar and cultured on potato dextrose agar. Living cultures were deposited in the RHS culture collection held at RHS Garden Wisley (Accession No. RHS454672) and at Westerdijk Fungal Biodiversity Institute, Netherlands (Accession No. CBS145765). The internal transcribed spacer (ITS) region of rDNA, the β-tubulin (Btub) gene and the translation elongation factor 1-alpha (EF1) gene were amplified using the primers ITS4:ITS5, T1:B-Sandy-R, and EF1-728F:EF2, respectively, according to the method by Verkley et al. (5). The DNA amplicons were sequenced (GenBank Accession Nos. MN093336 (ITS), MN105980 (Btub) and MN166626 (EF1)). The ITS sequence differed by one base pair from the only ITS sequence available for Septoria rudbeckiae (JQ677043). No previous sequences were available for comparison for Btub and EF1 and none of the available sequences had more than 90% identity. Pathogenicity was confirmed by spraying Rudbeckia fulgida sullivantii ‘Goldsturm’ plants with a conidial suspension (1 × 106 conidia/ml) prepared from spores from 21-day-old cultures on potato dextrose agar, incubated at 20°C with a 12 hr light/ 12 hr dark cycle. Plants were kept in high humidity for 72 hr after inoculation. After four weeks, black lesions were observed on leaves. Pycnidia and conidia consistent with S. rudbeckiae were found within each lesion. Control plants sprayed with sterilised water showed no symptoms. Septoria rudbeckiae was described from the USA (Ellis & Halsted, 1) where it is now widespread causing disfigurement of Rudbeckia in gardens. Although the fungus has been recorded from a number of different Rudbeckia species, Rudbeckia fulgida var. sullivanti cv. Goldstrum has been recognised in the USA as one of the most susceptible cultivars. Septoria rudbeckiae has also been reported from Canada, Bulgaria and Romania (Farr & Rossman, 2), Turkey (as Septoria sp.; Gumrukcu, 3) and Korea (Park, 4). To our knowledge, this is the first report of S. rudbeckiae in the United Kingdom. The authors would like to thank Jane Renshaw for aid in preparation of fungal cultures.

Daphne odora Thunb. an evergreen shrub with scented flowers, is used for ornamental purposes but it also has medicinal benefits (Otsuki, et al. 2020). In August 2021, leaf blotch symptoms were observed on roughly 20% of leaves of D. odora var. marginata plants in Fenghuangzhou Citizen Park, Nanchang city (28°41'48.12″ N, 115°52'40.47″ E), Jiangxi Province, China. Brown lesions first appeared on the edges of leaves, which eventually dried and died (Fig. 1A). For fungal isolation, 12 symptomatic leaves were randomly collected, the edges between diseased area and healthy area were cut into small pieces (4×4 mm), surface-sterilized by dipping in 70% ethanol for 10 s and 1% sodium hypochlorite for 30 s, and then rinsed three times with sterile distilled water. Leaf pieces were then plated on potato dextrose agar (PDA) and incubated at 28 ℃ for 3-4 days. A total of 10 isolates were recovered from the diseased leaves. The pure colonies of all fungal isolates had similar characteristics, and three isolates were randomly selected (JFRL 03-249, JFRL 03-250 and JFRL 03-251) for further study. Colonies of this fungus were gray and uneven, with a granular surface, and irregular white edges, finally turning black on PDA (Fig. 1B, C). Pycnidia were black, globose and 54-222 μm in diameter (Fig. 1D). Conidia were hyaline, single-celled, and nearly elliptical, which ranged from 7 to 13 × 5 to 7 μm (n=40) (Fig. 1E). These morphological characteristics were consistent with those described for the fungus Phyllosticta spp. (Wikee et al. 2013a). To confirm the fungal identity, the internal transcribed spacer (ITS) region, actin (ACT), translation elongation factor 1-alpha (TEF1-a), glyceradehyde-3-phosphate dehydrogenase (GPD) and RNA polymerase II second largest subunit (RPB2) genes were amplified using primers ITS5/ITS4, ACT-512F/ACT-783R, EF-728F/EF2, Gpd1-LM/Gpd2-LM and RPB2-5F2 /fRPB2-7cR, respectively (Wikee et al. 2013b). The sequences of the selected isolates were 100% identical. Hence, sequences of one representative isolate JFRL 03-250 were deposited in GenBank (OP854673, ITS; OP867004, ACT; OP867007, TEF1-a; OP867010, GPD; and OQ559562, RPB2). BLAST search analysis in GenBank showed 100% similarity with those of P. capitalensis (GenBank accession nos. ITS, MH183391; ACT, KY855662; TEF1-a, KM816635; GPD, OM640050 and RPB2, KY855820). From a phylogenetic perspective, a maximum likelihood phylogenetic tree was constructed by using IQtree V1.5.6 based on multiple sequences (ITS, ACT, TEF1-a, GPD and RPB2) (Nguyen et al. 2015), and the cluster analysis resulted the representative isolate JFRL 03-250 within a clade comprising Phyllosticta capitalensis (Fig. 2). Based on morphological and molecular characters, the isolate was identified as P. capitalensis. To confirm pathogenicity and fulfill Koch's postulates, 6 healthy potted plants were inoculated with 1× 106 conidia/ml suspension of isolate JFRL 03-250 by spraying on the leaves, whereas 6 plants were sprayed with sterile distilled water to serve as control. All potted plants were incubated at 28°C, 80% relative humidity and 12-h light/12-h dark alternating conditions in a climate cabinet. After 15 days, similar symptoms were observed in the inoculated leaves as in the field (Fig. 1F), whereas control leaves remained asymptomatic (Fig. 1G) and P. capitalensis was successfully re-isolated from the symptomatic leaves. Previously, P. capitalensis has been reported to cause brown leaf spot disease of various host plants around the world (Wikee et al. 2013b). However, to our knowledge, this is the first report of brown leaf spot caused by P. capitalensis on D. odora in China.

Ardisia crenata Sims, belonging to the family Myrsinaceae, has high medicinal and economic value. In July 2021,root rot disease was observed in plantations located in Ziyun (106°08'45″ E, 25°75'15″ N) and Xiuwen (106°46'53″ E, 26°54'01″ N) Counties, Guizhou Province, China, with an incidence rate of approximately 30% at fruit drop stage. The disease manifested as root softening, blackening, and phloem rot, while the aerial parts showed progressive yellowing, curling, and withering of leaves. Ultimately, the plant died. Nine symptomatic root segments were collected from fifteen infected plants, surface sterilized in 5% NaClO and 75% ethanol for 1 minute each, washed three times with sterile water, and incubated on potato dextrose agar (containing 10μg/mL of chloramphenicol) for five days at 28°C. The hyphal tip technique (Senanayake et al. 2020) was used to obtain pure cultures. Sixteen strains exhibiting similar morphological characteristics were isolated from the infected tissues. The colonies of these isolated cultures appeared white and turned light grey after 4 days. Five isolates were selected and grown on 2% water agar for 7 days for morphological characterization. Conidia were single-celled, hyaline, spindle-shaped to oval, and measured 12.04 to 19.47 µm long and 4.63 to 7.41 µm wide (n = 50). Based on these morphological features, the isolates were suspected to be Neofusicoccum parvum (Pavlic et al. 2009). For molecular identification, strain-7 was randomly selected as a representative individual to extract DNA. The internal transcribed spacer (ITS) region was amplified using primers ITS1/ITS4 (White et al. 1990). Additionally, the translation elongation factor 1-alpha (TEF1) gene was amplified using primers EF1-728F/EF1-986R (Rehner et al. 2005), and the β-tubulin (TUB2) gene was amplified using primers BT2a/BT2b (Glass et al. 1995). The sequences of strain-7 (accession number OR789487[ITS]; PV209690[TEF1]; PV209691[TUB2]) were deposited in GenBank, demonstrating a sequence homology of 99% to 100% (569/570, 265/265, 448/448) with N. parvum YBF5-1 (accession numbers PQ222752, PQ227810 and PQ227811). Based on morphological features and Neighbor-Joining method analyses of combined ITS, TEF1, and TUB2 gene sequences, strain-7 was identified as N. parvum. A pathogenicity test was performed using strain-7 by inoculating roots of 12-month-old A. crenata seedlings. The test was repeated seven times. Healthy seedlings were cut vertically with a sterile knife at 2 cm from the edge of one stem to create root damage. Spore suspensions (150 ml, 1 × 106 conidia/ml) of N. parvum were applied on the cut side, while the control group was watered with the same volume of sterile water. All plants were kept in the same glasshouse under natural conditions. After 7 days, some yellowish spots appeared on the leaf surface of some plants, with the edges turning dry and curled. The branches and leaves turned completely yellow, and the roots rotted extensively on the 14th day, whereas the control group remained asymptomatic. To satisfy Koch's postulates, N. parvum was reisolated from the inoculated plants but not from the control. N. parvum has been reported to cause leaf spot disease on Macadamia integrifolia and Vitis heyneanan in China (Li et al. 2023; Wu et al. 2015). This is the first report of N. parvum causing root rot in A. crenata in China. These findings provide a basis for the early detection of A. crenata root rot and the formulation of targeted control measures.

Goji berry (Lycium barbarum) is a plant of the Solanaceae family that is cultivated in the Chinese provinces of Xinjiang, Ningxia, Gansu, and Qinghai, and its fruit is used as a traditional Chinese medicine (Yossa Nzeuwa et al. 2019). In July 2019, fruit rot was observed at an incidence of 20 to 25% on the Goji berry at a fruit market in Yinchuan, Ningxia, China. The fruit symptoms began as slightly shriveled areas on fruit peel, with noticeable softening of the infested portion of the tissue, followed by rotting and a sour odor. To isolate the pathogen, ten symptomatic tissues were randomly collected from different boxes, surface-sterilized for 30 s with 75% ethanol, followed by 0.1% mercuric chloride, then rinsed in sterile distilled water three times and plated onto PDA. The plates were incubated at 25°C in the dark for 7 days. Five purified fungal isolates from different fruit were obtained and single-spores. Emergent fungal colonies were white with 1 to 3 mm white margins and abundant aerial hyphae, 1 to 6 mm high, that became dark gray after 4 to 5 days. Conidia were hyaline, unicellular, fusiform, and measured 19.3 to 28.2 μm× 3.8 to 6.4 μm (n=50). All the morphological characteristics were consistent with Botryosphaeria spp. (Slippers et al. 2004). Five representative isolates, BJN1-BJN5, were selected for molecular identification. Total genomic DNA of the isolates was extracted with a Plant/Fungi DNA Isolation Kit. Translation elongation factor 1-alpha (EF1) gene and internal transcribed spacer (ITS) regions were amplified with primers EF1-728F/986R (Carbone and Kohn 1999) and ITS1/ITS4, respectively. The sequencing results of the five isolates were consistent, and those of the isolate BJN1 we deposited in the NCBI GeneBank database for EF1 (MK733274) and ITS (MK359291). A BLAST search of the GenBank database indicated that the EF1 and ITS sequences had 100% and 99% similarity, respectively, to B. dothidea ex-type strain (AY236898 and KF766151). A phylogenetic tree was constructed using maximum parsimony methods in MEGA11 and BJN1 isolate clustered with the reference sequence of B. dothidea. Pathogenicity tests were performed, inoculating healthy fruit with both mycelial plugs (7 days old) and conidial suspension (1 × 106 conidia/ml), repeated three times. Mycelial plugs of five isolates (BJN1-BJN5) growing on PDA with a colony diameter of 4 mm were placed on the sterilized surface of 20 Goji berry fruit. Sterile PDA plugs were placed on 12 healthy fruit as a control. In a second test, conidial suspensions of five isolates were sprayed on the surface of 20 healthy fruit and sterilized distilled water was used as a control. The inoculated fruits were maintained in an artificial climate chamber at 25°C and 80% to 85% relative humidity with a 12-h photoperiod for 7 days. The development of soft rot, similar to that observed on the original samples, was observed on inoculated fruit while control fruits remained asymptomatic. The pathogen was reisolated from infected fruit and confirmed as B. dothidea based on morphological characteristics and molecular sequences. To our knowledge, this is the first report of B. dothidea causing postharvest fruit rot of Goji berry, and this pathogen has been reported to cause fruit rot in Kiwifruit (Li et al. 2016) and Yellowhorn (Liu et al. 2018). This study provides information on a new postharvest fruit rot of Goji berry in China that has the potential to cause economic losses.

In the summer of 2020, 127 soybean [Glycine max (L.) Merr] seedlings (V1-V3 stage) with reduced growth vigor were sampled as part of a bulk collection of seedling pathogens from Purdue's Agronomy Center for Research and Education in West Lafayette, Indiana. After rinsing off soil, one plant displayed prominent necrotic lesions on both cotyledons and the hypocotyl and rot of the roots. Root tissue segments measuring roughly 5 mm in length and adjacent to lesions were excised and surface sterilized in 0.6% NaOCl for 10 min, then in 70% ethanol for 2 min, rinsed thrice in sterile distilled H2O, and plated on dichloran-chloramphenicol-peptone agar (Andrews and Pitt 1986). Single-spore cultures were produced and grown on potato dextrose agar. The isolate (AC101) developed white aerial mycelium, rings of magenta coloration in the media, and pale orange sporodochia with age. Microscopic observation of two-week-old cultures grown on synthetic low-nutrient agar (NRRL Medium No. 4) in the dark at 28°C revealed 2-3 septate falcate macroconidia measuring 17.1 - 43.9 × 2.8 - 3.5 µm (avg. 29.4 × 3.1 µm, n=20); 0-1 septate straight to slightly curved microconidia measuring 3.9 - 8.6 × 1.9 - 2.5 µm (avg. 7.0 × 2.2 µm, n=20); and round chlamydospores borne singly or doubly with diameter measuring 6.1 - 14.2 µm (avg. 8.9 µm, n=20). These characteristics were consistent with descriptions of Fusarium commune K. Skovg., O'Donnell & Nirenberg (Skovgaard et al. 2003). DNA was extracted from aerial mycelium and the internal transcribed spacer (ITS) region using ITS1/ITS4 primers (White et al. 1990) (GenBank accession MW463361), the mitochondrial small subunit (mtSSU) rDNA using MS1/MS2 primers (White et al. 1990) (MW466537), and the translation elongation factor 1-alpha (TEF1α) gene using 983F/1567R primers (Rehner and Buckley 2005) (MW475296) were amplified and sequenced. Blast searches in GenBank showed that these sequences had 100% identity with corresponding sequences of F. commune (ITS: MN452698; mtSSU: AF362277; and TEF1α: KU171720). The matching mtSSU sequence was an accession from the original species description (Skovgaard et al. 2003). A pathogenicity test was conducted under greenhouse conditions (20-29°C, avg. 24°C) following the infested soil protocol of Ellis et al. (2013a). Ten seeds (cv. Williams) each were used in inoculated and mock-inoculated control treatments with one seed per foam cup. Root rot symptoms similar to, but more destructive than those observed in the field, were observed 14 days after planting on all inoculated plants but not on controls. Inoculated plants reached VE stage compared to controls which reached VC. Disease symptoms included severe necrotic lesions on the cotyledons, dark brown rot of the developing tap root, and brown hypocotyl lesions similar to field symptoms. F. commune was successfully reisolated from inoculated plants, but not from controls, as described above. F. commune has been reported to cause soybean root rot in China (Chang et al. 2018), Korea (Choi et al. 2020), as well as Iowa (Ellis et al. 2013b). To our knowledge this is the first report of F. commune infecting soybean seedlings in the state of Indiana. The expanded distribution of this soybean pathogen warrants heightened attention for its control.

Internal transcribed spacer or ITS region of nuclear ribosomal DNA (rDNA) has been used to evaluate genetic assortment and phylogenetic relationship in nine sugarcane genotypes including Saccharum species and another related genus as Erianthus, Narenga and hybrid. DNA was extracted from selected genotypes and ITS (ITS-1 and ITS-2) regions were amplified using specific primers. The sequence lengths ITS-1 showed 205- 207 bp, while ITS2 was ranged from 211- 218 bp. However, G+C content (%) 65.2% - 67% in ITS-1 and in ITS-2 68.4% - 99.7%. The sequence lengths of fragment and GC content of ITS-1 and ITS-2 regions showed variable. To evaluate the phylogenetic association of both the region of ITS (ITS-1 and ITS-2) neighbor-joining (NJ) method was employed. The cluster A of ITS-1 and cluster B for ITS-2 and cluster C combined between ITS1+ ITS2 sequences gave two distinct groups A and B. The group A represented the ITS1 sequences which showed two subgroups I and II. The A-I subgroup consisted of wild species of sugarcane; Erianthus, Narenga and S. robustum, whereas the A-II subgroup consisted of the Saccharum species and hybrid. The ITS2 sequences in the group B showed better correlation amongst each other. The sequences ITS-1 & ITS-2 combined and compared with some selected sequences from NCBI database using NJ method. The results have confirmed that ITS region can be used for evaluating the genetic assortment in Saccharum and its closely related genes.

Neonectria fuckeliana (C. Booth) Castl. & Rossman is the cause of nectria flute canker of exotic Pinus radiata plantations in New Zealand and Chile, where sunken cankers are associated with pruned branch stubs (Dick and Crane 2009; Morales 2009). The bright colored (red), superficial, aggregated perithecia are typical of the Nectriaceae (Hirooka et al. 2012). Nectria flute canker is a key biosecurity threat to Australia’s 1 million ha of exotic Pinus plantations, where annual surveys are conducted over the majority of the estate (Carnegie et al. 2008). During the course of forest health surveys in southeastern New South Wales, Australia, in October 2012, fruiting bodies typical of the Nectriaceae were detected on a dead P. radiata tree near Bombala. Teleomorphic structures were not observed, only anamorphic. Morphological features of the stromata (erumpent, orange to red), pycnidia (aggregated in groups of 3 to 10, superficial, subglobose, cerebriform to slightly cupulate, red to bay), and conidia (hyaline, ellipsoidal to oblong, nonseptate, 2.5 to 3.5 × 1 to 1.5 μm) corresponded to the description of Thyronectria pinicola (Kirschst.) Jaklitsch & Voglmayr (syn. Pleonectria pinicola) (Hirooka et al. 2012; Jaklitsch and Voglmayr 2014). Ascomata were selectively removed from bark with sterile forceps and DNA was extracted using the UltraClean Plant DNA Isolation Kit (MoBio Laboratories, Solana Beach, CA, USA). The internal transcribed spacer (ITS) and large subunit (LSU) regions of nuclear ribosomal DNA were amplified with ITS1F/ITS4 and LROR/LR7 as done by Hirooka et al. (2012). The ITS and LSU sequences were 100% identical to isolates of T. pinicola included in the studies by Hirooka et al. (2012). Specimens DAR 80239 (GenBank Accession Nos. ITS: KP751376, LSU: KP751378) and DAR 80240 (GenBank Accession Nos. ITS: KP751375, LSU: KP751377) were 100% identical to T. pinicola HM484540 (596/596 identities) and JF832615 (460/460 identities) in the ITS region, and 100% identical to HM484567 (801/801 identities) and JF832747 (795/795 identities) in the LSU region. Collections previously assigned to Nectria balsamea are now recognized as three distinct species that correlate to host plants: Thyronectria balsamea (Cooke & Peck) Jaklitsch & Voglmayr on Abies spp.; T. boothii (Hirooka, Rossman & P. Chaverri) Jaklitsch & Voglmayr on Picea spp.; and T. pinicola on Pinus spp. (Hirooka et al. 2012; Jaklitsch and Voglmayr 2014). T. pinicola is known from several Pinus spp. in the Northern Hemisphere, including Germany, Japan, and the USA, while T. balsamea is only known from Abies in North America and T. boothii from Picea in Slovakia (Hirooka et al. 2012). A search of Australian (http://www.planthealthaustralia.com.au) and New Zealand (http://nzfungi2.landcareresearch.co.nz) fungal herbaria indicates no previous records of T. pinicola in either of these countries, nor any nectriacous species on Pinus in Australia. This appears to be the first record of T. pinicola from Oceania, and also from P. radiata, the most important softwood plantation species in temperate regions of the Southern Hemisphere. Further surveys have revealed T. pinicola on fire-damaged and wind-blown P. radiata in several major pine-growing regions in New South Wales. Although T. pinicola is not considered a primary pathogen, this finding illustrates the benefit of forest health surveys in supplementing biosecurity surveys to detect biosecurity threats.

Acorus calamus var. angustatus Besser, a perennial herb of the Araceae family, was first reported in the ShenNong'sHerbalClassic and is widely distributed in southern China (Li 1979). It is important in traditional Chinese medicine for treating heart, stomach, and brain ailments (Lam et al. 2016). In March 2024, leaf spots were found on its leaves in a traditional Chinese medicine planting base in Yuexi County (30°91'56″ N, 116°19'24″ E), Anhui Province, with an incidence of about 35%. The symptoms began as small, light brown lesions that expanded, resulting in necrotic lesions ranging from 1 to 8 mm in diameter with brown halos. 3 × 3 mm sections, including both symptomatic and asymptomatic tissues, were cut from six infected plants. They were disinfected in 75% ethanol for 30 s, washed three times with sterile distilled water, transferred to Petri dishes containing potato dextrose agar (PDA) and incubated at 25 °C in the dark. Purified fungal isolates were obtained by the single-spore isolation method. The colonies on PDA initially appeared white, gradually became olive-green with 1 to 3 mm white margins and abundant aerial hyphae, while the reverse was greyish green to black. The conidia were light brown, ellipsoidal, obclavate, and 10.0 to 49.5 µm × 4.5 to 16.4 µm (mean 23.9 × 10.2 µm, n=50) in size, with 0 to 6 transverse septa and 0 to 6 longitudinal or oblique septa (n=50). Conidiophores were thick, dark brown, single-celled, with multiple conidial scars, measuring 12.8 to 146.8 × 2.9 to 6.1 (mean 50.6 × 4.2) µm (n=50). Based on above observations, the pathogen were identified as Alternaria spp. (Simmons 2007). Three representative isolates, SCP-1, SCP-2, and SCP-3 were selected for molecular identification. The Internal transcribed spacer (ITS), Alternaria major allergen (Alt a 1), and translation elongation factor 1-alpha (TEF1) genes were amplified with the primers ITS1/ITS4 (White et al. 1990), Alt-for/Alt-rev (Woudenberg et al. 2015) and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively. The sequences of the three isolates were consistent, and the sequences of isolate SCP-1 were submitted to NCBI GenBank (ITS, PP723104; Alt a1, PP708704; TEF1, PP708703). The ITS region of isolate SCP-1 was 100% similar to A. alternata TCS3002 (MN394880, Wang et al. 2023), the Alt a 1 gene was 100% similar to A. alternata CBS 620.83 (KP123868), and the TEF1 gene was 100% similar to A. alternata CBS 916.96 ex-type (KC584634). A phylogenetic tree based on sequences of ITS, Alt a 1 and TEF1 genes was constructed using the neighbor-joining method in MEGA 7 software, confirming the fungus as A. alternata. Three healthy plants of A. calamus var. angustatus were spayed with conidial suspension (1 × 107 conidia/ml) of isolate SCP-1. Three additional plants sprayed only with sterile distilled water were controls. All plants were covered with plastic bags to maintain a relative humidity of 90% for 48 h and incubated at 25 °C under a 12-h light photo-period. Twelve days post-inoculation, brown necrotic lesions developed on the inoculated leaves, enlarged, and the symptoms were similar to the original ones. The control plants remained healthy. The fungus was re-isolated from the infected plants and confirmed by morphological traits and molecular methods, fulfilling Koch's postulates. To our knowledge, there are no other reports of this fungus on A. calamus var. angustatus in Anhui, China. This report will help identify the disease based on field symptoms and provide a basis for disease management strategies of A. calamus var. angustatus.

Orthosiphon stamineus (Java tea) is a perennial herbaceous plant in the family Lamiaceae and is cultivated extensively in Southeast Asia for its medicinal value (Arifullah et al. 2014). During October 2018, leaf blight symptoms were observed on leaves of ~210 plants O. stamineus grown in experimental plots of a research farm at Faculty of Engineering, Serdang, Selangor, Malaysia (3°00'30.4"N 101°43'19.9"E) with 80% disease incidence. Initial symptoms were brown-to-black lesions on the leaves that enlarged and coalesced until the leaf withered and abscissed. Diseased tissues (4 × 4 mm) of six infected leaves were excised, surface disinfected with 0.5% NaOCl for 1 min, rinsed twice with sterile distilled water, placed onto potato dextrose agar (PDA) plates, and incubated at 25°C with a 12-h photoperiod under fluorescent light for 7 days. A total of six single-spore isolates were obtained from sampled leaves. All isolates exhibited similar morphology and two representative isolates, MK and MK1, were characterized further. Colonies on PDA were initially white then turned dark gray with age and had a pale green underside. Hyphae were branched, septate and hyaline to pale brown. The conidia were one-celled, black, smooth-walled, spherical to subspherical in shape measuring 11.0 μm × 16.5 μm in diameter (n=30) and which are borne on hyaline vesicles at the tip of each conidiophore or formed directly from the mycelia. Based on morphological characteristics, the fungal isolates were identified as Nigrospora osmanthi (Wang et al. 2017). Total genomic DNA of the isolates was extracted from fresh mycelium using DNeasy Plant Mini kit (Qiagen, USA) and the internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1) and Beta-tubulin (TUB2) gene regions were amplified using ITS5/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999) and Bt-2a/Bt-2b primer set (Glass and Donaldson 1995), respectively. BLASTn analysis showed that the ITS, TEF1 and TUB sequences of the isolates shared 99%-100% identity with Nigrospora osmanthi ex-type strain CGMCC 3.18126 (GenBank accession nos. KX986010, KY019421, KY019461). The sequences of two representative isolates (MK and MK1) were deposited in GenBank (ITS: accession nos. MT645782, MW363019; TEF1: MW366861, MW366862; TUB: MW366863, MW366864). Phylogenetic analysis by the maximum likelihood method based on the concatenated ITS-TEF1-TUB sequences showed that the isolates in this study were clustered in a strongly supported group 98% maximum likelihood with type strain N. osmanthi (Kumar et al. 2016). The pathogenicity of all isolates was confirmed by inoculation on ten healthy leaves of five potted 4-week-old O. stamineus plants using a conidial suspension (1 x 106 spores/ml) produced on 7-days-old PDA cultures. An equal number of plants were sprayed with sterile distilled water only to serve as a control and the treated plants were kept in a growth chamber for 2 weeks at 28 ± 1°C and 95% relative humidity. The experiment was repeated twice. The inoculated leaves developed brown lesions which enlarged into blight symptoms similar to those observed on naturally infected leaves after 5 days of inoculation, while control plants remained healthy. Nigrospora osmanthi was successfully re-isolated from the infected leaves, but not from leaves of non-inoculated control plants, thus satisfying Koch's postulates. . N. osmanthi has been recently reported to cause leaf blight on Ficus pandurata (Liu et al. 2019) and Stenotaphrum secundatum in China (Mei et al. 2019). This disease can cause a significant threat to the cultivation of O. stamineus which has been extensively grown for the production of herbal Java tea. Accurate identification of this pathogen could assist in developing an effective disease management strategy to control this disease.

Analysis of sequence data from the internal transcribed spacers (ITS) and 5.8S region of nuclear ribosomal DNA show that Canarian and Madeiran brooms (Genisteae) of the genera Teline, Adenocarpus, and Genista are related to Mediterranean species and not to species from adjacent parts of Morocco. Each separate colonization of the islands has resulted in contrasting patterns of adaptation and radiation. The genus Teline is polyphyletic, with both groups (the "T. monspessulana group" and the "T. linifolia group") separately nested within Genista. Genista benehoavensis (La Palma) and G. tenera (Madeira) form, with G. tinctoria of Europe, a single clade characterized by vestigially arillate seeds. The Canarian species of Adenocarpus have almost identical sequence to the Mediterranean A. complicatus and are likely to be the result of island speciation after a very recent colonization event. This Canarian/Mediterranean A. complicatus group is sister to the afrotropical montane A. mannii which is probably derived from an earlier colonization from the Mediterranean, possibly via the Red Sea hills. The independent colonization and subsequent radiation of the two Teline groups in the Canary Islands make an interesting comparison: the phylogenies both show geographical structuring, each with a central and western island division of taxa. Within the "T. monspessulana group" there is some evidence that both continental and Madeiran taxa could be derived from the Canary Islands, although it is likely that near contemporaneous speciation occurred via rapid colonization of the mainland and islands. The finding of two groups within Teline also has implications for patterns of hybridization in those parts of the world where Teline species are invasive; in California members of the T. monspessulana group hybridize readily, but no hybrids have been recorded with T. linifolia which has been introduced in the same areas.

In spring 2025, a nursery grower in Wimauma, FL, USA, submitted ‘Avignon Early Blue’ lavender (Lavandula angustifolia) plants (one-month-old, 1L pots) exhibiting stunted growth, leaf chlorosis and necrosis to the Plant Diagnostic Clinic at the University of Florida Gulf Coast Research and Education Center. Disease incidence on this sole cultivar was about 90% (n = ~1500) and severity was about 50%, distributed in three different areas of the nursery, under field conditions. Abundant sporangia and conidiophores that were characteristic of downy mildew pathogens were observed on the abaxial leaf surface. Severely affected leaves developed curling as necrosis progressed. Based on microscopy, the pathogen was identified as a downy mildew, evidenced by coenocytic mycelia dichotomously branched conidiophores, and sporangia. Conidiophores bearing sporangia were harvested from three separate plants (one from each infested area in the nursery) using a sterile needle, and DNA was extracted using the FastDNA kit (MP Biomedicals, Solon, OH). The internal transcribed spacer (ITS), translation elongation factor 1-alpha (EF1A), beta-tubulin (TUB), heat shock protein 90 (HSP90), and cytochrome c oxidase subunit I and ll (COX1, 2) were sequenced according to Hoffmeister et al. (2020). Sequences of the three isolates were deposited in GenBank (ITS: PV954822 to 24; COX1: PV995061 to 63; COX2: PV995064 to 66; EF1A: PV995067 to 69; TUB: PV995070 to 72; HSP90: PV995073 to 75). BLASTn searches revealed that isolates were 100% identical to Peronospora choii (holotype BPI 893223) accession numbers MN450333 (ITS, 1135/1135 bp), MN546902 (EF1A, 867/867 bp), MN546927 (TUB, 919/919 bp), MN547003 (HS90, 907/907 bp), MN546953 (679/679 bp), and MN546979 (496/496 bp). Phylogenetic analysis also revealed isolates grouped with Pe. choii. For morphological characterization, fifty conidiophores and fifty sporangia were collected, mounted on slides, and measured under the microscope. Conidiophores measured in length 363-798 μm (Average=420, Standard Deviation=5.3) and had submonopodial branching. Sporangia were greyish to pale brownish, ovoidal to ellipsoidal, and measured 13-25 μm long (Avg=21, SD=3.3), and 12-22 μm in width (Avg=19, SD=2.4). Oospores were not observed. For pathogenicity, ten ‘Avignon Early Blue’ plants were used, five for inoculation, and five as non-inoculated controls. Symptomatic tissues from the submitted samples that were frozen at –80°C were washed with deionized water containing 0.1% Tween 20 to release the sporangia, and the suspension was adjusted to 104 conidia/ml. Plants were spray inoculated until run-off (~20 ml/plant) and kept inside clear plastic boxes for 72 h. Control plants were sprayed with sterile deionized water plus 0.1% Tween 20. Afterward, plants were kept in a growth room (25°C and 12/12-h light/dark). The experiment was repeated once. Two weeks after inoculation, symptoms were observed in all inoculated plants with abundant sporangia and conidiophores production under the leaves, identical to the symptoms observed on the original sample. The non-inoculated controls remained healthy. To our knowledge, this is the first report worldwide of Pe. choii, a new species arising out of the Pe. belbahrii complex, causing downy mildew on lavender. Little is reported about downy mildew of lavender, but in Israel, it has been attributed to Pe. belbahrii (Ben-Naim et al., 2019; Thines et al., 2020). Peronospora choii is a new species formerly recognized in 2020 by Hoffmeister et al. (2020), which was found on Plectranthus scutellarioides in Tennessee (2015) and Michigan (2007), USA. If downy mildew of lavender caused by Pe. choii were to become a major problem, targeted management efforts such as cultivar screening, optimized fungicide applications, epidemiological research, and surveys of alternative hosts would be essential.