Exploring Factors Conditioning the Expression of Botryosphaeria Dieback in Grapevine for Integrated Management of the Disease.

Species from the Botryosphaeriaceae family are the causal agents of Botryosphaeria dieback (BD), a worldwide grapevine trunk disease. Because of their lifestyle and their adaptation to a wide range of temperatures, these fungi constitute a serious threat to vineyards and viticulture, especially in the actual context of climate change. Grapevine plants from both nurseries and vineyards are very susceptible to infections by botryosphaeriaceous fungi due to several cuts and wounds made during their propagation process and their entire life cycle, respectively. When decline becomes chronic or apoplectic, it reduces the longevity of the vineyard and affects the quality of the wine, leading to huge economic losses. Given the environmental impact of fungicides, and their short period of effectiveness in protecting pruning wounds, alternative strategies are being developed to fight BD fungal pathogens and limit their propagation. Among them, biological control has been recognized as a promising and sustainable alternative. However, there is still no effective strategy for combating this complex disease, conditioned by both fungal life traits and host tolerance traits, in relationships with the whole microbiome/microbiota. To provide sound guidance for an effective and sustainable integrated management of BD, by combining the limitation of infection risk, tolerant grapevine cultivars, and biological control, this review explores some of the factors conditioning the expression of BD in grapevine. Among them, the lifestyle of BD-associated pathogens, their pathogenicity factors, the cultivar traits of tolerance or susceptibility, and the biocontrol potential of Bacillus spp. and Trichoderma spp. are discussed.

First Report of Neofusicoccum parvum causing leaf spot on Camellia japonica in China.

Camellia japonica is an important garden landscape plant in southern China. In April 2022, leaf spot symptoms were observed at the camellia garden of Jiaying University (24°32'83″N, 17 116°12'31″E) in Meizhou city, Guangdong Province, China. The initial symptoms were grayish brown spots on the leaves, as the disease progressed, the lesions were enlarged and affected the whole leaf and eventually led to the loss of its ornamental value. The disease incidence was above 15%. Leaf pieces (5 × 5 mm) from 3 diseased Camellia leaves were sterilized in 75% ethanol for 1 min, then in 1% NaOCl for 1 min; and rinsed three times with sterile water. Leaf pieces were inoculated on potato dextrose agar (PDA) medium and incubated at 25 °C. Three days later, fungal colonies initially showed a white aerial mycelium, turning gray after 5 days, and dark gray after 7 days of incubation. Conidia were single-celled, hyaline, ellipsoidal and without septa. Dimensions of conidia (n≥50) were 14.27 to 20.65 × 4.28 to 6.56 µm. The morphological characteristics matched the genus Neofusicoccum (Pavlic et al. 2009). For molecular identification, the rDNA internal transcribed spacer (ITS1, 5.8S and ITS2) region, translation elongation factor 1-alpha (tef1-α), and beta-tubulin (tub2) of a representative isolate SC6-2 were amplified using the primer pairs ITS1/ITS4, EF1/EF2 and BT2a/-BT2b, respectively (Golzar and Burgess，2011). The sequences obtained were deposited in GenBank (accession nos. PP064173, PP479650 and PP082457 for ITS, tef1-α and tub2, respectively). Nucleotide BLAST analysis showed a 99.81% homology with N. parvum (519/520 bp, OQ509869; 519/520 bp, KF294003; 518/519 bp KF293989) for ITS, 100% homology with N. parvum (398/398 bp, MN318108; 398/398 bp, MK294085; 398/398 bp, MH936021) for tub2, and >99% homology with N. parvum (259/259 bp, 100%, MW390561; 263/265 bp, 99.25%，MN175952; 263/265 bp, 99.25%, MK781982) for tef1-α. The combined phylogenetic analyses (ITS, tef1-α, and tub2) showed that the sequence of the tested isolate and the corresponding sequence of N. parvum (CMW9081, SHSJ1-2) in GenBank grouped in the same branch of the phylogenetic tree. Based on morphological characters, DNA sequencing, and the phylogenetic tree, it can be determined that the pathogen was Neofusicoccum parvum. Inoculation on Camellia leaves was performed to confirm pathogenicity. Nine healthy camellia leaves were pin-pricked with a sterile needle and inoculated with mycelial plugs of isolate SC6-2. Nine other healthy leaves were pin-pricked and inoculated with noncolonized PDA plugs as control leaves. The inoculated leaves were maintained on water agar solid medium at 25°C. To keep a high-humidity environment the inoculation sites were covered by moistened cotton for 2 days. The experiment was repeated three times. Five days after inoculation, all the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 6 days. The fungal isolates recovered from inoculated leaves were morphologically identical to the N. parvum isolates originally recovered from symptomatic leaves collected in the field, fulfilling Koch's postulates. Neofusicoccum parvum is an aggressive pathogen that causes severe disease on important tree and woody species (Liddle et al. 2019). It has been reported that N. parvum can infect the leaves and branches of grapes (Otoya-Martinez et al. 2023), dieback on Camellia japonica (Pintos, et al. 2012), Brazilian pepperwood (Bertetti et al. 2022), mango (Giancarlo et al. 2023) and other plants. To our knowledge, this is the first report of N. parvum causing leaf spot on Camellia japonica in China.

First Report of Neofusicoccum parvum Causing Seedpod Blight on Lotus (Nelumbo nucifera) in China.

Lotus (Nelumbo nucifera Gaertn.) is a widely cultivated plant in China, and the fruit lotus variety has a high economic value attributed to the exquisite flavor of its fresh seeds. During the summer of 2023, an unidentified blight was observed affecting lotus seedpods in Jiande City, Zhejiang province, with approximately 65% of seedpods impacted in a 130-hectare area. The initial symptoms included dark purple spots on the lotus seedpod surface, which gradually expanded over time. After 5 to 7 days, the entire seedpod turned black, withering, and rendering the lotus seeds inedible. To identify the causal agent, tissues from symptomatic seedpods were excised and disinfected in 75% ethanol for 60 s, and washed twice in sterile distilled water. The disinfected symptomatic tissues (5 × 5 mm) were plated on potato dextrose agar (PDA), incubated at 25 ℃, transferred hyphal tips to obtain pure isolates after 3 days. Fungal colonies exhibiting Botryosphaeriaceae morphology were isolated from 33% of the samples (n = 15). Pure cultures were grown on PDA for both morphological and molecular identification. The colonies displayed a white aerial mycelium, turning olivaceous grey after 7 days. Pycnidia were produced within 3 weeks on PDA with added sterilized healthy lotus seedpod pieces on the surface. Conidia were hyaline, unicellular, ellipsoidal, 12.65 to 20.72 × 3.92 to 9.38 µm in size (mean 16.67 × 6.24 µm, n = 100). To determine the fungal species, genomic DNA was extracted from one representative isolate (ZJUP1112-1), to amplify four gene loci through polymerase chain reactions (PCR): rDNA internal transcribed spacer (ITS) with primers ITS1/ITS4, rDNA large subunit (LSU) with LR0R/LR5, the translation elongation factor 1-alpha gene (tef1) with EF1-728F/EF1-986R, and ß-tubulin gene (tub2) with Bt2a/Bt2b. The PCR products were Sanger sequenced in Zhejiang Shangya biotechnology co., LTD, and the resulting sequences were assembled and deposited in GenBank (ITS: OR740546; LSU: OR740547; tef1: OR776996; tub2: OR776997). BLAST searches indicated the highest nucleotide sequence identity with the reference strains of Neofusicoccum parvum CMW 9081 (ITS: 98.8%, AY236943; LSU: 100%, AY928045; tef1: 99.6%, AY236888; tub2: 99.3%, AY236917). Multi-locus phylogenetic analyses revealed that isolate ZJUP1112-1 formed a highly supported clade with N. parvum. Pathogenicity tests were performed on healthy lotus seedpods using mycelial plugs (5 mm diameter) from actively growing colonies of ZJUP1112-1 that were placed onto the front and side of the seedpods (6 each). Controls received PDA plugs. Treated seedpods were wrapped with parafilm and incubated at 25 ℃ and the experiment was repeated three times. After 5 days, dark purple lesions were observed on the inoculated seedpods, whereas controls remained symptomless. The same isolate was recovered from the margin of resulting lesions and confirmed by morphology, thus fulfilling Koch's postulates. N. parvum is a polyphagous pathogen causing blights and fruit rot on multiple economically important fruit crops, such as cacao (Puig et al. 2019), walnut (Chen et al. 2019), pistachio (Lopez-Moral et al. 2020), chestnut (Seddaiu et al. 2021), blueberry (Spetik et al. 2023) and mango (Polizzi et al. 2022), among others. To the best of our knowledge, this is the first report of N. parvum causing seedpod blight on lotus seedpods in China, which contributes to a better understanding of the pathogens affecting this plant species in China.

First Report of Postharvest Fruit Rot Caused by Neofusicoccum parvum on Prunus salicina (Nai Plum) in China.

Nai plum (Prunus salicina var. cordata cv. Younai) is one of the most popular fruit crop in South China. In July 2023, a fruit rot of nai plum with about 5 % disease incidence was observed in a fruit market of Changsha city, Hunan Province, China. Initially, small, brown lesions appeared randomly on the fruit surface, with disease progression, the lesions gradually expanded and developed into soft rot. To isolate possible fungi from rotten fruits, small pieces (2 × 2 mm) from the periphery of 10 infected fruits were surface-sterilized using 70% ethanol for 10 s, rinsed three times in sterile distilled water, air dried, and then placed onto potato dextrose agar (PDA) plates and incubated at 28℃ for three days. Emerging colonies were subcultured by hyphal tip transfer on fresh PDA. A total of ten isolates with similar morphology were obtained. Fungal colonies were initially white, gradually turning gray and eventually becoming black, and aerial hyphae were dense and fluffy. Conidia were hyaline, single celled, ellipsoidal to fusiform, and range from 12.7 to 20.0 µm long (avg. 16.9 ± 2.39 µm) × 5.3 to 7.3 µm wide (avg. 6.3 ± 0.82 µm). These morphological characteristics of these isolates matched those of Neofusicoccum parvum (Phillips et al. 2013). To future confirmation of the identify, the internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (TEF1-a), and beta-tubulin TUB2) genes of two representative isolates (JXNP1 and JXNP2) were amplified and sequenced using primer sets ITS5/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999; Phillips et al. 2013), and BT2A/BT2B (Glass and Donaldson 1995), respectively. The sequences of both isolates were deposited in GenBank for the ITS (accession nos. OR899331 and OR899332), TEF1-a gene (accession nos. OR909890 and OR909891) and TUB2 gene (accession nos. OR909892 and OR909893). BLAST analysis showed 99-100% identity with the ex-type strain of N. parvum (CMW9081) for ITS, TEF1-a and TUB2. A maximum likelihood phylogenetic tree was constructed using IQtree web server based on combined ITS, TEF1-a and TUB2 data set. The phylogenetic tree revealed that two isolates clustered with N. parvum in a clade with 90% bootstrap support. Based on morphological and molecular data analysis, the isolates were identified as N. parvum. To confirm the pathogenicity, five healthy nai plum fruits were wounded by using a sterile needle after surface sterilization with 75% ethanol, then a 5-mm-diameter mycelial disc of isolate JXNP1 was taped to the wound, the control fruits were taped with sterile agar plugs. All fruits were incubated at 25 ℃ with 80% humidity. After five days, typical naturally occurring fruit rot symptoms appeared on the fruits which inoculated with N. parvum, whereas control fruits remained asymptomatic. To fulfill Koch's postulates, the pathogen was re-isolated from the inoculated fruits and comfirmed as N. parvum by morphological and molecular analysis. Previous studies reported that N. parvum caused fruit rot on various common fruits in China, including loquat, kiwifruit and citrus (Lei et al. 2013; Zhai et al. 2019; Zhou et al. 2013). To our knowledge, this is the first report of N. parvum causing postharvest fruit rot on nai plum in China. This finding provides critical insights for the management of the high-risk disease on plum in China.

First Report of Neofusicoccum parvum Causing Fruit Rot on Psidium guajava in China.

Guava (Psidium guajava L.), a nutrient-rich and economically significant fruit, is extensively cultivated in southern China. In six continuous years (from 2018 to 2023), dark-purple rotted guava with sunken lesion were observed on guava trees (variety 'Zhenzhu', aged over 5 years) in Dongguan and Panyu districts, Guangdong Province. Annually, the incidence of fruit rot disease in the affected fields reached 30% to 50% and significantly reduced the yield and quality of harvest guava. The initial symptoms on the epicarp of the fruits were black, needle-like dots that rapidly spread, causing partial or complete fruit rot within two to three weeks. To identify the causative agent, six symptomatic fruits were collected from two different orchards. Samples of 0.5 cm³ were excised from the lesion margins of each fruit. These samples were surface-sterilized with 70% ethanol for 30 seconds, followed by 0.2% NaClO for 2-3 minutes, and rinsed in sterile water three times. The samples were then cultured on potato dextrose agar (PDA) at 25°C for five days. This process yielded eight fungal isolates with similar morphological. Initially, the colonies were white with dense aerial mycelium becoming dark gray after 4 to 5 days. The mycelia were septate and branched. No spores were observed on PDA. To induce spore formation, the isolates were cultured on water agar for 20 days. This process led to the production of hyaline, aseptate, ellipsoid conidia, which were thin-walled, smooth-surfaced, and measured 3.7-5.1 × 1.6-2.2 µm (n = 100). Three isolates, including at least one from each orchard (Np1, Np2, Np3), were selected for further analysis. Genomic DNA was extracted using Axygen MAG-FRAG-I-50 (Axygen Bio-Tek). The internal transcribed spacer of rDNAs (ITS), beta-tubulin (tub2), the nuclear ribosomal large subunit (LSU), and translation elongation factor 1-α (tef1-α) gene regions were amplified using the primers ITS1/4, Bt2a/Bt2b, LR5/LR0R, and EF1-728F/EF1-986R (Golzar and Burgess 2011) and sequenced. Sequence analysis using MEGA 7.0 (Kumar et al., 2018) revealed 100% similarity among the isolates. BLAST searches of the ITS, tub2, LSU, and tef1-α sequences (accession nos. MN907637, MT537938, MT528156, MT537939) showed the highest nucleotide similarities (99.24 to 100%) to Neofusicoccum parvum strains (Crous et al. 2006). A phylogenetic tree was constructed with MEGA 7.0 based on the nucleotide sequence tub2 using the maximum likelihood method. Pathogenicity tests on 10 healthy guava fruits using mycelium-inoculated and control fruits confirmed the causative agent. The inoculated fruits, maintained at 25°C under a 12-h light/dark cycle, exhibited symptoms identical to the field infections within four to seven days, while control fruits remained symptomless. The fungus, reisolated from the inoculated fruits, was morphologically identical to the original isolates, fulfilling Koch's postulates. In conclusion, based on molecular, morphological, and pathogenic analysis, N. parvum as the causal agent of the fruit rot disease on guava. Previously, N. parvum has been reported in association with fruit rot on Eriobotrya japonica and Juglans regia (Zhai et al. 2019; Chen et al. 2019). To our knowledge, this is the first report of N. parvum affecting guava in China.

The fungal metabolite 4-hydroxyphenylacetic acid from Neofusicoccum parvum modulates defence responses in grapevine.

In a consequence of global warming, grapevine trunk diseases (GTDs) have become a pertinent problem to viticulture, because endophytic fungi can turn necrotrophic upon host stress killing the plant. In Neofusicoccum parvum Bt-67, plant-derived ferulic acid makes the fungus release Fusicoccin aglycone triggering plant cell death. Now, we show that the absence of ferulic acid lets the fungus secrete 4-hydroxyphenylacetic acid (4-HPA), mimicking the effect of auxins on grapevine defence and facilitating fungal spread. Using Vitis suspension cells, we dissected the mode of action of 4-HPA during defence triggered by the bacterial cell-death elicitor, harpin. Early responses (cytoskeletal remodelling and calcium influx) are inhibited, as well as the expression of Stilbene Synthase 27 and phytoalexin accumulation. In contrast to other auxins, 4-HPA quells transcripts for the auxin conjugating GRETCHEN HAGEN 3. We suggest that 4-HPA is a key component of the endophytic phase of N. parvum Bt-67 preventing host cell death. Therefore, our study paves the way to understand how GTDs regulate their latent phase for successful colonisation, before turning necrotrophic and killing the vines.

First report of Neofusicoccum parvum causing stem blight and dieback of highbush blueberry in the Czech Republic.

Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips is a cosmopolitan pathogen causing dieback of multiple diverse woody hosts including highbush blueberry (Vaccinium corymbosum L.). This fungus can survive inside colonized plants without causing any symptoms for several years. Once the endophytic lifestyle is switched to the parasitic one, the symptoms of dieback can rapidly occur (bronze leaves, necroses under the bark, apoplexy) and the plant usually declines within a few weeks (Slipper and Wingfield 2007). In August 2022, blueberry plants displaying symptoms described above were observed in a production orchard located in Hovorany, the Czech Republic. Around 3 % of 1000 observed plants were symptomatic. In order to identify the pathogen, leaves, stems and roots of three diseased plants were collected, sectioned into small pieces (5 × 5 mm), surface sterilized (60 s in 75% ethanol, followed by 60 s in 1% sodium hypochlorite and rinsed three times using sterile distilled water), plated on potato dextrose agar (PDA) supplemented with 0.5 g/liter of streptomycin sulfate (PDAS) (Biosynth, Staad, Switzerland) and incubated at 25°C for 2 weeks at dark. Newly developed mycelia were immediately transferred to fresh PDA plates and purified by single-spore or hyphal-tip isolation. In total 33 fungal isolates were obtained. All the 33 isolates shared similar morphology and resembled Botryosphaeriaceae spp. Colonies on PDA (7 d at 25°C) were felty, white to iron grey in the centre. Conidiomata were observed on sterile pine needles on 2 % water agar (WA) at 25°C under near-UV light after 2 wks (110-220 × 60-175 µm). Conidia (n=30) were cylindrical to ellipsoidal, hyaline, 0(-1)-septate, (3.8-8.1 × 2-3 µm). Two representative isolates were deposited at the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands (CBS 149846 and CBS 149847). The partial internal transcribed spacer (ITS) regions, beta-tubulin gene (tub2) and translation elongation factor 1-alpha (tef) gene were amplified from genomic DNA of both isolates following primers and protocols previously described (Eichmeier et al. 2020). Newly generated sequences were deposited in NCBI GenBank (acc. nos. ITS: OQ376566, OQ376567; tub2: OQ401701, OQ401702 and tef: OQ401699, OQ401700), being >99% identical (ITS 483/484 nt, tub2 426/430 nt and tef 230/232 nt) with the ex-type ITS (AY236943), tub2 (AY236888) and tef (AY236917) sequences of N. parvum strain CMW9081. Phylogenetically, newly obtained isolates grouped with ex-type and another three cultures of N. parvum in the three gene-based phylogenetic tree with strong 98/1.0 (BP/PP) support. To confirm pathogenicity, one-year-old canes of ten two-year-old V. corymbosum plants grown in pots were wounded by a 5 mm diam cork borer, and a 5-mm mycelial plug of a 7-day-old culture of both (CBS 149846 and CBS 149847) strains (five plants per strain) was inserted into the wound. Ten plants were inoculated with sterile PDA plugs and served as controls. Wounds were covered by sterile wet cotton, sealed with Parafilm® and inoculated plants were maintained in a growth chamber at 20 °C with 12/12 h light/dark period. Within two weeks, inoculated shoots changed colour from green to dark brown and exhibited dark necroses under the bark; after one month inoculated plants declined, while controls remained symptomless. The pathogen was reisolated from the inoculated plants with 100 % re-isolation rate, and its identity confirmed by sequencing ITS region. The experiment was repeated. Neofusicoccum parvum causing dieback of highbush blueberry was already reported from Australia, California, Chile, China, Italy, Mexico, Portugal and Uruguay (Rossman et al. 2023). Pecenka et al. (2021) reported a presence of another pathogen - Lasiodiplodia theobromae (Pat.) Griffon & Maubl. from the same plantation. This suggests that stem blight and dieback of highbush blueberry is caused by more than one Botryosphaeriaceae spp. as it was previously proved by Xu et al. (2015). To the best of our knowledge, this is the first report of stem blight and dieback of highbush blueberry caused by N. parvum in the Czech Republic.

Natamycin Has an Inhibitory Effect on Neofusicoccum parvum, the Pathogen of Chestnuts.

This research aimed to investigate natamycin's antifungal effect and its mechanism against the chestnut pathogen Neofusicoccum parvum. Natamycin's inhibitory effects on N. parvum were investigated using a drug-containing plate culture method and an in vivo assay in chestnuts and shell buckets. The antifungal mechanism of action of natamycin on N. parvum was investigated by conducting staining experiments of the fungal cell wall and cell membrane. Natamycin had a minimum inhibitory concentration (MIC) of 100 µg/mL and a minimum fungicidal concentration (MFC) of 200 µg/mL against N. parvum. At five times the MFC, natamycin had a strong antifungal effect on chestnuts in vivo, and it effectively reduced morbidity and extended the storage period. The cell membrane was the primary target of natamycin action against N. parvum. Natamycin inhibits ergosterol synthesis, disrupts cell membranes, and causes intracellular protein, nucleic acid, and other macromolecule leakages. Furthermore, natamycin can cause oxidative damage to the fungus, as evidenced by decreased superoxide dismutase and catalase enzyme activity. Natamycin exerts a strong antifungal effect on the pathogenic fungus N. parvum from chestnuts, mainly through the disruption of fungal cell membranes.

First Report of Brown Leaf Spot Caused by Neofusicoccum parvum on Photinia × fraseri Dress (Christmas berry) in China.

Christmas berry (Photinia × fraseri Dress), known as red tip photinia, is an important landscape color plant in South China (Zhu et al., 2021). In summer 2021, brown leaf spots of Christmas berry were observed with an incidence of 45-70% on the campus of Nanchang Institute of Technology, Jiangxi Province (28°41'32.61"N, 116°1'53.75"E), China. These spots have a negative influence on the photosynthetic activity and severely reduce the ornamental value of these plants. Lesions originally occurred on the margins or tips of the leaves as red patches (Figure 1 A) and then developed into irregular reddish-brown necrotic spots with yellow halos (Figure 1 B) that finally were grey lesions with dark brown ring circles surround with yellow halos (Figure 1 C). Ten leaf samples with typical symptoms from five plants were collected. The tissue between the healthy and necrotic area (ca. 5 mm × 5 mm) was cut with a sterile scalpel and surface sterilized in 70% alcohol for 45 s, followed by 2% NaClO for 2 min and washed in sterile deionized water three times, The sterilized leaf parts were placed on potato dextrose agar (PDA) and incubated at 25℃ for 3 to 5 days. A total of 28 fungal isolates were obtained from ten symptomatic leaves, which were classified into five species by morphological characteristics. Neofusicoccum was the genus with the highest isolation frequency (50%). For pathogenicity tests, healthy leaves of Christmas berry were surface-sterilized, wounded using a sterile needle and inoculated with mycelial plugs (6 mm in diameter) on the left site of the leaves. Five representative isolates were chosen and ten leaves were used for every isolate. The controls on the same leaves on the right side of the leaves were treated with sterile PDA plugs. All samples were placed in an artificial climate box (RH 90±3%, 27±1℃, 12 h light) for 3 days. Only the leaves inoculated with isolate NH7 showed dark brown leaf spots (Figure 1 D). The control leaves and those inoculated with the other four isolates remained symptomless. To confirm pathogenicity, mycelial plugs of isolate NH7 were applied on punctured leaves of Christmas berry in the field as the artificial climate box (Figure 1 E-H). Inoculation with a sterile PDA plug served as control. All the leaves were covered with plastic bags for 48 h to maintain high relative humidity. Seven days later, symptoms similar to those observed in the field developed on all leaves inoculated with isolated NH7, while the controls remained symptomless. To fulfill Koch's postulates, fungal isolates were reisolated from symptomatic leaves and identified by morphological and molecular characteristics. The colony of isolate NH7 developed aerial hyphae, which had a grey center surrounded by grey-white hyphae (Figure 1 I). Conidia were aseptate, primarily fusiform and measured 11.1-19.8 × 3.2-7.1 µm (n = 50) (Figure 1 J). For further confirmation of the identity, five genes, including ITS (White et al., 1990), RPB2 (Pavlic et al., 2009), tub2 (Glass & Donaldson, 1995), and tef1 (Carbone & Kohn, 1999) were sequenced. The sequences were deposited in GenBank (OL584411 for ITS, OL606622 for ACT, OL694623 for tub2, OM141481 for RPB2 and OM141482 for tef1), Based on the phylogenetic tree analysis using IQ-TREE, isolate NH7 clustered with Neofusicoccum parvum (Figure 2). N. parvum has been reported to cause leaf spot disease on different plants including Myristica fragrans (Jayakumar et al., 2011), Ginkgo biloba (Mirhosseini et al., 2014), Vitis heyneana (Wu et al., 2015), rubber tree (Liu et al., 2017) and Geodorum eulophioides (Du et al., 2021). To our knowledge, this is the first report of N. parvum causing leaf spot disease on Christmas berry in China. As one of the most widely planted ornamental shrubs in China, the detailed knowledge of the pathogens targeting Christmas berry is critical.

First report of seedling stem blight of mango caused by Neofusicoccum parvum in Italy.

Seedling blight of mango (Mangifera indica L.) was observed in a commercial nursery located in Messina province (eastern Sicily, Italy) during winter of 2021. More than 30% of 3,000 seedlings, about three to six months old, of mango cv. Gomera 3 showed symptoms of basal stem blight. The symptoms started from seed, led to the decline and subsequent death of the plants. Necrotic lesions appeared at crown level two months after sowing. The stem tissues of ten symptomatic plants were cut, surface sterilized, dipped in 1.5% sodium hypochlorite for 1 min and transferred onto potato dextrose agar medium (PDA) and incubated at 25°C for four days. Approximately 60% of stem tissues developed very similar fungal colonies, resembling to Botryosphaeriaceae. A total of four representative isolates were collected through single hyphal-tip and stored at 4 °C. The internal transcriber spacer region (ITS) was amplified with primers, ITS5/ITS4 (White et al., 1990), and EF1-728F and EF1-986R (Carbone and Kohn, 1999) were used to amplify part of the translation elongation factor 1alpha gene (tef1-α), and primers Bt2a/Bt2b (Glass and Donaldson, 1995) were used for the partial ß-tubulin (tub2). The obtained sequences were deposited in GenBank with accession numbers: ON911292-95 for the ITS, ON933621-24 for tef1-α and ON933625-28 for tub2.To compare the results, 50 additional sequences were selected and inserted in the alignment according to the recent literature on the Botryosphaeriaceae (Bezerra et al., 2021; Zhang et al., 2021). Maximum parsimony analysis (MP) of concatenated dataset (ITS + tef1-α + tub2) was performed in PAUP v.4.0a. Clade support was assessed by 1,000 bootstrap replicates and Botryosphaeria dothidea was used as an outgroup. Our isolates clustered within the group of Neofusicoccum parvum (71% bootstrap value) (ex-type CMW9081). Based on these results, and morphological data (50 conidia length × width average: 18.1 × 6.6, respectively) our isolates (named MC) were identified and confirmed as Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips. Pathogenicity tests were also conducted on 18 mango cv. Gomera 3 seedlings. The crown roots of each seedling were mechanically wounded and a mycelial plug of the isolate MC14 was placed onto them and covered with soil. Controls (three seedlings) were inoculated with sterile PDA only. Seedlings were maintained in a growth chamber with a 12 hrs photoperiod at 25°C ± 1°C and watered regularly. After five days, stem lesions appeared externally (1.6 cm) and one month after the inoculation, all the inoculated seedlings died. However, controls did not show any obvious symptoms. Re-isolations were conducted as described above and fulfilled Koch's postulates confirming pathogenicity. Among the diseases affecting mango plants, Botryosphaeriaceae represent a serious threat in Sicily as reported by Aiello et al., 2022. The endophytic behaviour of Botryosphaeriaceae is well known, making them latent pathogens (Slippers and Wingfield, 2007). In Italy, N. parvum was detected in mango orchards since 2013 (Ismail et al. 2013), but symptoms of seedlings stem blight have never been reported in the nursery. In Sicily, an increase of Botryosphaeriaceae infection has been observed recently, especially in nurseries, where N. parvum has been identified as a most destructive pathogen (Aiello et al., 2020; Gusella et al., 2021). To our knowledge, this is the first report worldwide of N. parvum causing mango seedling blight. The high incidence of infected seedlings detected in this study highlights the potential risk during propagation in the nursery, representing a significant source of inoculum for the field.

First Report of Neofusicoccum parvum Causing Leaf Spot on coral trees (Viburnum odoratissimum) in China.

Coral trees (Viburnum odoratissimum), as a class of evergreen shrubs, are mainly planted in landscapes in numerous cities in China. During September 2020, the author investigated four major parks in Hefei (Bao Park, Hefei Botanical Garden, Luzhou Park and Peninsula Park) and the campus of Anhui Agricultural University (approximately 0.5 ha) (31°49'21.30″N, 117°13'18.25″E). The results showed that the incidence rate of leaf spot disease reached 60% among approximately 100,000 coral trees planted in these areas. Coral trees begin to show leaf spots in August. In early stages of coral trees infection, the symptoms appeared as small brown spots ranged in length from 2 to 3 millimeters on the leaves. After the disease patches expand and darken, the coral leaves eventually wither and fall, which seriously affects its viewing and admiring value. To identify the fungal pathogen, the five-point sampling method was used to take typical similar leaf samples from 5 regions, and 6 samples were taken from each site, so a total of 150 samples were obtained. Fragments of sample leaves were surface-sterilized with 1% NaClO, plated on potato dextrose agar, and incubated at 25 °C in the dark. A total of 275 strains were obtained from 150 samples. According to the morphological characteristics, 275 strains were purified and divided into four types. Four representative strains (MI1, K1, F1, D1) were selected from four types for further pathogenicity testing and identification. The pathogenicity test was conducted in triplicate by inoculating wounded leaves of 1-year-old potted V. odoratissimum with 20µL of a conidial suspension (106 conidia/mL). The control was inoculated with sterile water. The specimens were placed in a growth chamber while maintaining 90% relative humidity and 28℃. After five days, the characteristic lesions were observed only on inoculated MI1 spore suspension leaves. The same fungus was reisolated from the lesions, thus fulfilling Koch's postulates. The pathogenic fungi accounted for 60% of all strains. Fungal colonies were circular and had abundant white aerial mycelium, and colonies changed from white to pure black after maturity. Conidia were fusiform (16-17×5-6 µm), thin-walled, transparent, and without diaphragms. Molecular identification was performed by partially sequencing the internal transcribed spacer (ITS) gene, the translation elongation Factor 1-alpha(EF1-alpha) gene, and the ß-tubulin (TUB2) gene by using the primers ITS1/ITS4 (White et al. 1990), EF1-728F (Alves et al. 2008)/EF1-986R (Carbone & Kohn 1999), and Bt2a/Bt2b (Glass & Donaldson 1995), respectively. The obtained ITS sequence (MW767713) showed 99% identity with N. parvum CMW28429 (KU997429.1), the EF1-alpha sequence (MZ398261) showed 99% identity with N. parvum isolate A4 (FJ528597.1), and the TUB2 sequence (MZ398260) showed 99% identity with N. parvum isolate BO52 (KU554657.1). By combining the sequences of individual fragments of each fungus in the order ITS, EF1-alpha and TUB2, MEGA 6.0 was used to analyze the sequence of kinship by using the maximum likelihood method, and the repeat value of bootstraps was 1000. A polygenic phylogenetic tree analysis based on multilocus alignment (ITS, EF1-alpha and TUB2) was constructed with some strains of Botryosphaeriaceae species. The results of the phylogenetic tree showed that MI1 and N. parvum clustered into a branch. To our knowledge, this is the first report of N. parvum causing leaf spot on V. odoratissimum in China.

First report of Neofusicoccum parvum causing leaf spot disease on Macadamia integrifolia in China.

The macadamia industry is developing rapidly in China. A brown leaf spot disease was noted in six Macadamia integrifolia plantations in Lincang, Yunnan, in October 2021. Over 60% of trees sampled had brown leaf spot symptoms, among approx.15,000 trees planted in these areas. Lesions (3 to 5 mm dia.) were small round brown spots with yellow edges. Lesions on severely infected leaves were darker and larger, with irregular shape (8 to 10 mm long, 3 to 6 mm wide). About 10% of diseased leaves had lesions characterized by a shot hole surrounded by a yellow halo. Potential pathogens were isolated from four randomly-selected symptomatic leaves from each of the six plantations by cutting lesion edges into small pieces. The pieces were surface sterilized, placed onto water agar containing 100 ppm aureomycin and incubated for 5 days at 24°C in the dark. Subculturing microbial growth on potato dextrose agar produced single-hyphal isolates with white fluffy aerial mycelia that turned pale olivaceous gray after 4 to 5 days. In four randomly-selected cultures, conidia were single celled, hyaline, spindle shaped to oval, and measured 10.9 to 16.3 µm long and 4.0 to 6.2 µm wide (n = 50). These characteristics matched those of Neofusicoccum parvum (Pavlic et al. 2009). Isolate LC013 was randomly selected as a representative individual for molecular identification. Internal transcribed spacer (ITS; ITS1/ITS4 primers; White et al. 1990), beta-tubulin gene (tub2; BT2A/BT2B primers; Glass and Donaldson 1995) and translation elongation factor 1-alpha gene (tef1-α; EF1-728F/EF2 primers; Carbone and Kohn 1999; O'Donnell et al. 1998) regions were PCR amplified from genomic DNA. Sequences of the products were used to BLAST probe the type specimen nucleotide sequences in GenBank. The LC013 sequences (GenBank accessions OM392021 (ITS); OM453641 (tub2); OM567656 (tef1-α)) had >99% sequence identity with analogous sequences from the type specimen of N. parvum CBS 138823 (accessions AY236943 (ITS); AY236917 (tub2); AY236888 (tef1-α)). Isolate LC013 was sister to N. parvum type strain in a maximum-likelihood (ML) tree constructed from analogous concatenated ITS, tef1-α, and tub2 sequences of 27 species that are phylogenetically closely-related to LC013 based on the ITS single locus ML tree. Koch's postulates were tested twice with two isolates by wounding leaves of four 14-month-old M. integrifolia seedlings with a sterile needle and placing a 5-mm-diameter agar plug containing N. parvum on the wound site. PDA plugs alone were used as uninoculated controls. Leaves were covered with sealed bags to maintain >90% humidity for 24 hours. All plants were kept in the same glasshouse under natural conditions. Leaves of inoculated plants began to discolor at 5 days post-inoculation (dpi). Brown spot symptoms were observed at 9 dpi. Control plants were symptomless. N. parvum was re-isolated from leaf lesions of the infected plants, but not from control plants, thus fulfilling Koch's postulates. N. parvum is an aggressive pathogen that causes severe disease on important tree and woody species, including M. integrifolia (Liddle et al. 2019). In China, it has been reported to cause leaf spot disease on 26 plant species (Farr and Rossman 2022), but this is the first report of N. parvum causing leaf spot disease on M. integrifolia. Further investigation is required to estimate the importance of this pathogen to the macadamia industry in China.

Effects of Cultivar Susceptibility, Branch Age, and Temperature on Infection by Botryosphaeriaceae and Diaporthe Fungi on English Walnut (Juglans regia).

Botryosphaeriaceae and Diaporthe fungi have been described as the main causal agents of branch dieback and shoot blight of English walnut (Juglans regia L.). To date, the effects of biotic and abiotic factors on disease development on this host are still poorly understood. Thus, the main goal of this study was to evaluate the effects of cultivar, shoot-branch age, and temperature on infection by Botryosphaeriaceae and Diaporthe fungi on English walnut. The susceptibility of eight commercial cultivars was evaluated against three Botryosphaeriaceae and two Diaporthe species. For the remaining experiments, shoots or branches of 'Chandler' were used. An initial experiment evaluating two inoculation methods was conducted, with inoculation with a mycelial plug being more consistent and useful than conidial suspension inoculation. Cultivar susceptibility varied depending on the fungal species, with 'Chandler' being among the most tolerant cultivars for shoot infection. One-year-old shoots were significantly more sensitive for both Neofusicoccum parvum and Diaporthe neotheicola in comparison with 2- to 4-year-old branches. The effect of temperature on shoot infection was evaluated under 5, 10, 15, 20, 25, 30, and 35°C. Lesion development was significantly higher for N. parvum isolates than for D. neotheicola isolates at all temperatures evaluated, with optimum temperature of shoot infection being ∼26°C for N. parvum and ∼21°C for D. neotheicola.

Tandem Mass Tags Quantitative Proteome Identification and Function Analysis of ABC Transporters in Neofusicoccum parvum.

Neofusicoccum parvum can cause twig blight of the walnut (Juglans spp.), resulting in great economic losses and ecological damage. We performed proteomic tandem mass tags (TMT) quantification of two Neofusicoccum parvum strains with different substrates, BH01 in walnut substrate (SW) and sterile water (SK), and BH03 in walnut substrate (WW) and sterile water (WK), in order to identify differentially expressed proteins. We identified 998, 95, and 489 differentially expressed proteins (DEPs) between the SK vs. WK, SW vs. SK, and WW vs. WK comparison groups, respectively. A phylogenetic analysis was performed to classify the ABC transporter proteins annotated in the TMT protein quantification into eight groups. Physicochemical and structural analyses of the 24 ATP-binding cassette (ABC) transporter proteins revealed that 14 of them had transmembrane structures. To elucidate the functions of these transmembrane proteins, we determined the relative expression levels of ABC transporter genes in strains cultured in sodium chloride, hydrogen peroxide, copper sulfate, and carbendazim mediums, in comparison with pure medium; analysis revealed differential upregulation. To verify the expression results, we knocked out the NpABC2 gene and compared the wild-type and knockout mutant strains. The knockout mutant strains exhibited a higher sensitivity to antifungal drugs. Furthermore, the virulence of the knockout mutant strains was significantly lower than the wild-type strains, thus implying that NpABC2 plays a role in the drug resistance of N. parvum and affects its virulence.

Ancestral chemotypes of cultivated grapevine with resistance to Botryosphaeriaceae-related dieback allocate metabolism towards bioactive stilbenes.

Grapevine trunk diseases have devastating consequences on vineyards worldwide. European wild grapevines (Vitis vinifera subs. sylvestris) from the last viable population in Germany along the Rhine river showed variable degrees of resistance against Neofusicoccum parvum (strain Bt-67), a fungus associated with Botryosphaeriaceae-related dieback. Representative genotypes from different subclades of this population were mapped with respect to their ability to induce wood necrosis, as well as their defence responses in a controlled inoculation system. The difference in colonization patterns could be confirmed by cryo-scanning electron microscopy, while there was no relationship between vessel diameter and infection success. Resistant lines accumulated more stilbenes, that were in addition significantly partitioned to nonglycosylated viniferin trimers. By contrast, the susceptible genotypes accumulated less stilbenes with a significantly higher proportion of glycosylated piceid. We suggest a model in which in the resistant genotypes phenylpropanoid metabolism is channelled rapidly and specifically to the bioactive stilbenes. Our study specifies a resistant chemotype against grapevines trunk diseases and paves a way to breed for resistance against grapevine Botryosphaeriaceae-related dieback.

Neofusicoccum parvum Causing Stem and Branch Blight Disease of Zanthoxylum bungeanum in Sichuan, China.

The Chinese pepper Zanthoxylum bungeanum Maxim is a special economically important species and a traditional spice in China. It is widely used in medicine, food, timber, tourism, soil and water conservation. In April 2019, A stem and branch blight disease of Z. bungeanum was discovered in Muli, Puge and Yanyuan counties, in Liangshan Prefecture (27°15'20″-27°19'38″N, 101°44'58″-102°04'10″E), causing approximately 15% yield loss in the three counties. Among all fields in Muli County, approximately 41.38%, 10.79% and 2% of Chinese peppers exhibited mild, moderate and severe branch blight, respectively. The symptoms started to occur from March to April. First, red-brown spots on the base of the stem, branches or main trunks of young trees observed but were not obvious. In May, the spots became gray-brown to dark brown ovals and gradually expanded into long strips (Figure 1a, b). When the spots surrounded the branches, the branches above them withered and died, and the spots gradually expanded downward. Around June or July, scattered black dot-shaped fruiting bodies were observed on the lesion. The branches of infected trees were sampled systematically by cutting the branch at the junction of infected and healthy areas in 5×5 mm sections. Each sample was surface-sterilized with 3% NaClO and 75% alcohol for 60 s before being rinsed three times with sterilized distilled water. The sterile filter paper was used to dry the tissue, and the samples were cultured on potato dextrose agar (PDA) amended with streptomycin sulfate (50 µg/ml). Plates were incubated at 25°C in the dark. From the five isolates obtained, four exhibited the morphology described by Yu et al. (2015) for Neofusicoccum parvum. The colonies were white fluffy at first and grew fast (Figure 1c). After five days, the colony diameter reached 75.2-84.8 mm, produced yellow pigment and the mycelium in the middle of the colony began to turn gray (Figure 1d). and the entire colony turned dark gray 7-8 days post culturing as observed previously (Javier-Alva et al. 2009) and formed a black fruiting body at 20 days (Figure 1e). The width of the mycelium measured 2.3-4.8 µm, and with the diaphragm (Figure 1f). The spores were round or fusiform, colorless, transparent, smooth, thin-walled, and measured 6.3-10.6×3.1-5.2 µm (Figure 1g, h), similar to N. parvum (Yu et al. 2013). For molecular identification, DNA was extracted from the mycelia of four fungal isolates using a plant genomic DNA extraction kit (Solarbio, Beijing). Polymerase Chain Reaction (PCR) was performed with the primers ITS1/ITS4 (White et al. 1990), EF446F/EF1035R (Inderbitzin et al. 2005), BTF/BTR2 and HspF3/HspR (Inderbitzin et al. 2010) for the ribosomal internal transcribed spacer region (ITS), elongation factor-1alpha (EF1-alpha), beta-tubulin (TUB) and heat shock protein (HSP) genes, respectively. BLAST searches in the GenBank database indicated that the ITS, TUB, HSP and EF-1α sequences had 100%, 99.0%, 99.7% and 99.7% identity to N. parvum, respectively. Representative sequences were deposited in GenBank (ITS: MT355871; TUB: MT409397; EF-1α: MT409399; HSP: MT460413). A pathogenicity test was performed using N. parvum on ten 2-year-old potted Z. bungeanum plants at 22-28°C and 60% humidity indoors. The conidial suspension (1×107 conidia/ml) collected 25 days old PDA cultures with 0.05% tween buffer was used for inoculation by brushing the wounded area of branch scratched by epidermis with a piece of sandpaper. Ten plants in pots were inoculated with sterile water and served as controls. Thirty days post-inoculation, the plants showed the same symptoms as the original diseased plants, and the controls remained asymptomatic. N. parvum was re-isolated from the infected tissues and identified by morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times with similar results, confirming Koch's postulates. This fungus is an important pathogen on a variety of woody hosts, and represents a serious problem in the vineyards worldwide (Mélanie, et al. 2017). To our knowledge, this is the first report of N. parvum causing stem and branch blight of Z. bungeanum trees in China.

Identification and Pathogenicity of Diplodia, Neofusicoccum, Cadophora, and Diaporthe Species Associated with Cordon Dieback in Kiwifruit cultivar Hayward in Central Chile.

Dieback symptoms associated with fungal trunk pathogens cause significant economic losses for farmers of kiwifruit and other woody fruit trees worldwide. This study represents the first attempt to identify and characterize the fungal trunk pathogens associated with cordon dieback disease of kiwifruit in central Chile. Field surveys were conducted throughout the main kiwifruit-growing regions in central Chile to determine the incidence and characterize the fungal trunk pathogens associated with cordon dieback of kiwifruit cultivar Hayward through morphological, molecular, and pathogenicity studies. A total of 250 cordon samples were collected and isolations were performed on 2% acidified potato dextrose agar (APDA) plus antibiotics and Igepal. The incidence of kiwifruit cordon dieback ranged between 5% and 85% in all surveyed areas in central Chile. A total of 246 isolates were isolated and identified using culture and morphological features belonging to three fungal taxa: Diaporthaceae spp. (Diaporthe ambigua and D. australafricana; n = 133 isolates); Botryosphaeriaceae spp. (Diplodia seriata and Neofusicoccum parvum; n = 89 isolates); and Ploettnerulaceae spp. (Cadophora luteo-olivacea and C. malorum; n = 24 isolates). These were identified using phylogenetics studies of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) of the rDNA, part of the ß-tubulin gene (tub2), and part of the translation elongation factor 1-α gene (tef1-α). Isolates of N. parvum and D. seriata were the most virulent, causing internal brown lesions and dieback symptoms in attached green shoots, attached lignified canes, and young inoculated kiwifruits. This report is the first to describe D. seriata and C. luteo-olivacea associated with kiwifruit cordon dieback in Chile. It presents the first description of N. parvum causing kiwifruit dieback worldwide.

First Report of Neofusicoccum parvum Causing Leaf Spot on Geodorum eulophioides in China.

Geodorum eulophioides Schltr. is a critically endangered orchid listed in the International Union for Conservation of Nature (IUCN) Red List of threatened species. At present, only two natural populations were found in China. It has important scientific and ornamental values because of its uniqueness. During the summer of 2019, a black leaf spot disease occurred on G. eulophioides, in Yachang Orchid National Nature Reserve (E106°13'32″，N24°44'19″) in Guangxi province, China. More than 60% of leaves of these plants were infected. The disease symptoms initially appeared as small yellow circular spots, which enlarged into irregular brown spots (6 to 9 cm length and 3 to 5 cm width). In later stages of the disease development, the center of the spots became dark brown with a clear edge and surrounded by a yellow halo. In severe infections, the spots coalesced covering the entire leaf. Six symptomatic leaves were collected from three infected plants, surface sterilized in 75% ethanol for 15 s and 0.1% HgCl2 for 4 min, and subsequently washed three times with sterile water, then plated onto potato dextrose agar (PDA), and incubated at 28℃ for three days. Eighteen fungal cultures with similar morphological characteristics were obtained from the infected tissues. Colonies were initially white, then turned dark grey after nine days. To induce sporulation, isolates were grown on 2% water agar and incubated under UVA light at 28℃ for nine days. Three isolates were selected for morphological characterization. Conidia were hyaline, unicellular, nonseptate, ellipsoidal to fusiform, externally smooth, thin-walled, and ranged from 10.7 to 16.6 µm (avg. 13.8 µm) × 4.1 to 6.7 µm (avg. 5.1 µm) (n=50). The isolate DBL-1 was selected as a representative for molecular identification. Genomic DNA was extracted and used for PCR to amplify the rDNA internal transcribed spacer region (ITS), translation elongation factor 1-alpha gene (EF1-α), and beta-tubulin gene (TUB2), using the primer pairs ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R（Alves et al. 2008；Carbone & Kohn, 1999）, and T1/T2 (O'Donnell et al., 1997), respectively. The obtained ITS sequence (GenBank Accession No. MN918440), EF1-α sequence (MN963815), and TUB2 sequence (MN963816) showed >99% homology with several GenBank sequences of Neofusicoccum parvum (JX513636, KU997497 for ITS, KU997261, MH252401 for EF1-α, and KJ841779, MK412882 for TUB2, respectively). Based on morphological characteristics of the asexual morph and maximum likelihood analyses of a combined rDNA-ITS, EF1-α and TUB2 gene sequences, was identified as N. parvum. Pathogenicity test was performed using isolate DBL-1 by inoculating 3 leaves of G. eulophioides plants. The test was repeated three times. Each leaf was wounded using a sterile needle, and a mycelial plug (6 mm diameter) harvested from the periphery of a 3-day-old colony grown on PDA was placed on each wound. Plants were then covered with plastic bags to maintain high relative humidity of 90% and kept at 28℃ in a greenhouse under natural daylight conditions. An equal number of leaves on the same plant were inoculated using sterile PDA plugs and served as mock inoculated controls. After three days, all the inoculated leaves showed black spot symptoms resembling those observed in the field, whereas controls remained symptomless. The fungus was re-isolated from the symptomatic leaves, thus completing Koch's postulates. N. parvum has been reported to cause leaf spot disease on Myristica fragrans (Jayakumar, et al., 2011), Ginkgo biloba (Mirhosseini, et al., 2014), Vitis heyneana (Wu, et al., 2015), and Hevea brasiliensis (Liu et al., 2017), respectively. To the best of our knowledge, this is the first report of N. parvum causing leaf spot disease on G. eulophioides in China. The disease control measures and in-situ conservation method need to be strengthened to protect this rare species.