Neofusicoccum Parvum Research Articles

First report of almond decline syndrome caused by Neofusicoccum parvum in Italy

Almond (Prunus dulcis Mill. D. A. Webb) is considered a major crop in the Mediterranean countries. In Italy, almond orchards cover an area of 54,939 ha, with a yield of 82,146 t, with the cultivation widely distributed across several areas, such as southern regions. During the summer of 2023, 3-year-old almond plants cultivated in a commercial orchard in southern Piedmont (44°32’41.92’’ N; 7°57’25.7’’ E) showed wilting and sudden decline of the whole plant with an incidence of 15%. Main symptoms were cankers on the trunk characterized by a chocolate-brown discoloration of wood, cambium and even bark and occasional presence of gummy exudate. Symptomatic bark samples were rinsed with water, then blotted dry, and briefly soaked in ethanol for 15 s. Small pieces (5 × 5 mm) from the margin of necrotic areas were cut and placed on potato dextrose agar (PDA) plates amended with streptomycin sulphate (150 μg/ml). After incubation in the dark at 25 °C for 4 days fungal colonies emerging from the plated fragments were subcultured as monohyphal fungal cultures. They were fast-growing, turning from withish to smoky grey or grey-olivaceous after a week. Stromatic conidiomata were produced in pine needle agar, with pycnidia forming hyaline, globose to ellipsoidal, unicellular conidia, ranging in size from 16.9–17.3 μm × 5.4–5.6 μm. The causal agent was preliminary identified as Neofusicoccum sp. based on morphology. Two representative isolates (MTB3, MTC4) were identified by sequencing the ITS region and translation elongation factor 1-alpha (TEF1-α) gene with the primer pairs ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999). The sequences of the two isolates were deposited in NCBI GenBank with accession numbers PP930646 / PP930647 (ITS), and PP933185 / PP933186 (TEF-1α). BLAST analysis revealed 99% similarity with reference sequences of Neofusicoccum parvum isolate CMW9081. A maximum-likelihood phylogenetic tree was constructed using MEGA7 software based on the combined ITS and TEF1-α dataset. The phylogenetic tree demonstrated that the two isolates grouped with N. parvum in a clade with the ex-type CMW9081. The pathogenicity of both isolates was evaluated on 1-year-old ‘Vairo’ almond potted plants grafted on GF677 rootstock, to assess Koch’s postulate. For the inoculation, 5 mm in diameter mycelium plugs taken from a 7-days-old PDA culture were placed on the surface of fresh wounds made with a sterile cork-borer (5 mm-diameter) on the stem. Control plants were inoculated with sterile PDA plugs. The test was conducted twice. Three blocks of three plants per isolate and control were arranged in a randomized block design. All inoculated plants showed symptoms of shoot blight and cankers 2-weeks after inoculation. Additionally, gumming, pycnidia production and internal wood discoloration developed on the inoculation sites, with lesion length spreading for 16.8 ± 1.6 and 15.4 ± 3.0 cm for MTB3 and MTC4, respectively. Most of the plants died one month after inoculation. Control plants showed no symptoms. The pathogen was consistently reisolated from inoculated plants and the identity was confirmed through TEF-1α sequencing. Thus, results of Koch postulate clearly identify N. parvum as the etiological agent of the observed disease of almond in Piedmont. N. parvum was recently reported in the same area on hazelnut (Waqas et al. 2021) and blueberry (Guarnaccia et al. 2021), and as causal agent of band canker and branch dieback in almond trees in US and Spain (Moral et al. 2019). To our knowledge, this is the first report of N. parvum as the causal agent of almond decline syndrome in Italy. Considering the increasing of almond cultivation in Italy, this disease represents a threat for the future production.

Effect of arbuscular mycorrhizal symbiosis on grapevine response to Neofusicoccum parvum, a major trunk disease fungus

Grapevine is an economically-important culture worldwide but is currently the target of decline, especially caused by grapevine trunk diseases (GTD). One of the major grapevine trunk disease is Botryosphaeria dieback, which is associated with Botryosphaeriaceae fungi. Among the different methods that could contribute to increase grapevine fitness under stresses, viticulture could take advantage of symbiosis with arbuscular mycorrhizal fungi. Within this context, we investigated the effect of V. vinifera cv. Gewurztraminer colonization with the arbuscular mycorrhizal fungus Rhizophagus irregularis on plant tolerance to wood inoculation with Neofusicoccum parvum Bt67, one of the most aggressive fungus associated with Botryosphaeria dieback. We showed that grapevine mycorrhization resulted in a small but significant reduction of wood necrosis size and less intense necrosis symptoms on the leaves. We further characterized the response of grapevine leaves and roots to both arbuscular mycorrhizal fungus (AMF) symbiosis and GTD fungus inoculation, especially the interaction between these two conditions, with a non-targeted metabolomic approach. In the roots, both mycorrhization and N. parvum infection triggered metabolite reprogramming, especially sugars and stilbenes, which were downregulated by both AMF symbiosis and pathogen infection. Furthermore, N. parvum infection triggered a significant decrease in fatty acids and oxylipins in leaves of non-mycorrhized plants, whereas contents were maintained or increased in Rhizophagus irregularis-colonized plants. In conclusion, AMF symbiosis may be an interesting tool to improve health of young grapevines and help sustaining infection by trunk disease fungi, by harnessing lipid metabolism.

Genetic diversity and population structure of Botryosphaeria dothidea and Neofusicoccum parvum on English walnut (Juglans regia L.) in France

Botryosphaeriaceae species are the major causal agents of walnut dieback worldwide, along with Diaporthe species. Botryosphaeria dothidea and Neofusicoccum parvum are the only two Botryosphaeriaceae species associated with this recently emergent disease in France, and little is known about their diversity, structure, origin and dispersion in French walnut orchards. A total of 381 isolates of both species were genetically typed using a sequence-based microsatellite genotyping (SSR-seq) method. This analysis revealed a low genetic diversity and a high clonality of these populations, in agreement with their clonal mode of reproduction. The genetic similarity among populations, regardless of the tissue type and the presence of symptoms, supports the hypothesis that these pathogens can move between fruits and twigs and display latent pathogen lifestyles. Contrasting genetic patterns between N. parvum populations from Californian and Spanish walnut orchards and the French ones suggested no conclusive evidence for pathogen transmission from infected materials. The high genetic similarity with French vineyards populations suggested instead putative transmission between these hosts, which was also observed with B. dothidea populations. Overall, this study provides critical insight into the epidemiology of two important pathogens involved in the emerging dieback of French walnut orchards, including their distribution, potential to mate, putative origin and disease pathways.

Isolation of Antagonistic Endophytic Fungi from Postharvest Chestnuts and Their Biocontrol on Host Fungal Pathogens.

In this study, antagonistic endophytic fungi were isolated from postharvest chestnut fruits; endophytic antagonistic fungi and their combination of inhibitory effects on the fungal pathogen Neofusicoccum parvum were evaluated. A total of 612 endophytic fungi were isolated from 300 healthy chestnut kernels, and 6 strains out of them including NS-3, NS-11, NS-38, NS-43, NS-56, and NS-58 were confirmed as antagonistic endophytic fungi against Neofusicoccum parvum; these were separately identified as Penicillium chermesinum, Penicillium italicum, Penicillium decaturense, Penicillium oxalicum, Talarmyces siamensis, and Penicillium guanacastense. Some mixed antagonistic endophytic fungi, such as NS-3-38, NS-11-38, NS-43-56, and NS-56-58-38, exhibited a much stronger antifungal activity against N. parvum than that applied individually. Among them, the mixture of NS-3-38 showed the highest antifungal activity, and the inhibition rate was up to 86.67%. The fermentation broth of NS-3, NS-38, and their combinations exhibited an obvious antifungal activity against N. parvum, and the ethyl acetate phase extract of NS-3-38 had the strongest antifungal activity, for which the inhibitory rate was up to 90.19%. The NS-3-38 fermentation broth combined with a chitosan coating significantly reduced N. parvum incidence in chestnuts from 100% to 19%. Furthermore, the fruit decay and weight loss of chestnuts during storage were significantly decreased by the NS-3-38 fermentation broth mixture along with a chitosan coating. Therefore, a mixture of P. chermesinum and P. decaturense could be used as a potential complex biocontrol agent to control postharvest fruit decay in chestnuts.

Enhancing epidemiological knowledge of Botryosphaeriaceae in Mexican vineyards

Grapevine cultivation in Mexico is important, especially in the states of Baja California and Coahuila, which are the main wine production regions in the country. Grapevine trunk diseases (GTDs) impact productivity and cause substantial economic losses, with Botryosphaeria dieback being one of the most destructive. This disease is caused by fungi in the Botryosphaeriaceae, including species of Botryosphaeria, Diplodia, Lasiodiplodia, and Neofusicoccum. To date, Lasiodiplodia spp. are the primary Botryosphaeriaceae fungi reported in Mexico. The present study aimed to enhance the epidemiological knowledge of Botryosphaeriaceae in Mexican vineyards. Samples from grapevine plants exhibiting disease symptoms were collected from the states of Baja California and Coahuila. Of a total of 37 Botryosphaeriaceae isolates, six species were identified: Neofusicoccum parvum, N. australe, N. vitifusiforme, Botryosphaeria dothidea, Diplodia corticola, and D. seriata. Neofusicoccum parvum isolates were the most virulent, but were less virulent than previously reported Lasiodiplodia spp. The optimum growth temperatures for N. parvum and B. dothidea were from 28 to 30°C, but 25°C for D. seriata, N. vitifusiforme, and N. australe isolates. Only D. seriata isolates recovered growth when transferred to room temperature after exposure to 37°C or 40°C. This report is the first identification of B. dothidea and N. parvum as causative agents of Botryosphaeria dieback in the vine-growing regions of Mexico.

Diversity and Pathogenicity of Botryosphaeriaceae Species Isolated from Olives in Istria, Croatia, and Evaluation of Varietal Resistance.

During 2021 and 2022, a field investigation was conducted in Istria, Croatia, searching for trees exhibiting signs of Botryosphaeria dieback. Samples of symptomatic trees were collected from 26 different locations and analysed. Isolates that morphologically corresponded to species from the Botryosphaeriaceae family were selected, and detailed morphological characterisation and molecular identification of the isolates were conducted. Based on morphological characteristics and phylogenetic analysis using the internal transcribed spacer (ITS), beta-tubulin (TUB2), and translation elongation factor 1-alpha (TEF1-α) regions, six species of fungi from the Botryosphaeriaceae family were identified: Botryosphaeria dothidea (Moug. ex Fr.) Ces. & De Not.; Diplodia mutila (Fr.) Fr.; Diplodia seriata De Not.; Dothiorella iberica A.J.L. Phillips, J. Luque & A. Alves; Dothiorella sarmentorum (Fr.) A.J.L. Phillips, Alves & Luque; and Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips. This is the first report of D. mutila, Do. sarmentorum, and Do. iberica causing Botryosphaeria dieback on olive trees in Croatia, and the first study investigating the resistance of Croatian olive varieties to species from the Botryosphaeriaceae family. Pathogenicity testing of selected isolates and assessment of variety resistance were conducted on four different olive varieties, namely Buža, Istarska bjelica, Leccino, and Rosinjola, using representative isolates of the mentioned species. The most aggressive species was found to be N. parvum. Olive varieties exhibited differences in susceptibility depending on the fungus they were infected with.

Botryosphaeriaceae partially overlap on asymptomatic and symptomatic tissues of Anacardiaceae in agroecosystems and conservation areas in northern South Africa.

Members of the Botryosphaeriaceae are well-known endophytes and stress-related pathogens. We recently characterised the diversity of Botryosphaeriaceae in healthy tissues of three tree species in the Anacardiaceae, namely Sclerocarya birrea, Mangifera indica and Lannea schweinfurthii. Here we ask how that diversity compares with the Botryosphaeriaceae diversity associated with dieback on those tree species. Samples were collected from agroecosystems (Tshikundamalema and Tshipise in Limpopo) and conservation areas (Nwanedi and the Mapungubwe National Park in Limpopo and the Kruger National Park in Mpumalanga) ecosystems. Species were characterised using multigene sequence data and morphological data. Diplodia allocellula, Dothiorella brevicollis, Do. viticola, Lasiodiplodia crassispora, L. mahajangana and Neofusicoccum parvum occurred on both asymptomatic and symptomatic samples. Dothiorella dulcispinea, L. gonubiensis and L. exigua, as well as a previously unknown species described here as Oblongocollomyces ednahkunjekuae sp. nov, only occurred in asymptomatic branches. An interesting aspect of the biology of O. ednahkunjekuaeae is that it appears to be adapted to higher temperatures, with an optimum growth at 30 °C, and faster growth at 35 °C than at 25 °C. Lasiodiplodia pseudotheobromae only occurred in symptomatic branches. Neofusicoccum parvum was notably absent from conservation areas, and in agroecosystem it was most common on M. indica. Only L. crassispora and L. mahajangana overlapped on all three tree species and were the dominant species associated with dieback. These results show that not all Botryosphaeriaceae occurring asymptomatically in an area contribute equally to disease development on a related group of hosts, and that environmental disturbance plays a significant role in the distribution of N. parvum. Citation: Slippers B, Ramabulana E, Coetzee MPA (2024). Botryosphaeriaceae partially overlap on asymptomatic and symptomatic tissues of Anacardiaceae in agroecosystems and conservation areas in northern South Africa. Fungal Systematics and Evolution 13: 131-142. doi: 10.3114/fuse.2024.13.07.

Chitosan and GRAS Substances: An Alternative for the Control of Neofusicoccum parvum In Vitro, Elicitor and Maintenance of the Postharvest Quality of Avocado Fruits

Chitosan and GRAS Substances: An Alternative for the Control of Neofusicoccum parvum In Vitro, Elicitor and Maintenance of the Postharvest Quality of Avocado Fruits

Emerging pathogens and disease dynamics threatening avocado production in southern Türkiye

AbstractAvocado (Persea americana Milll.) holds a pivotal position in global fruit crops, contributing significantly to the economies of tropical and subtropical regions. However, the rising incidence of diseases poses a substantial risk to avocado production. This comprehensive study investigated the disease landscape in Antalya, the largest avocado cultivation area in the Türkiye. A survey of 2537 avocado trees across 11 regions from 2020 to 2021 revealed alarming disease incidences, particularly in the eastern regions of Gazipasa and Alanya. Dieback, branch canker, anthracnose, and soil-borne root rot were identified as the primary diseases affecting tree canopies, twigs, and branches. Morphological and molecular analyzes unveiled a spectrum of pathogens, with Colletotrichum gloeosporioides dominating in the Mediterranean region. Notably, Phytophthora cinnamomi emerged as a severe threat, causing root rot and decline in avocado trees. Fusarium solani and Fusarium oxysporum, known for their association with tropical fruit crops, were identified in the western parts of Antalya. Additionally, we have detected Neofusicoccum parvum, Lasiodiplodia theobromae, and Neopestalotiopsis rosae in collected samples from avocado trees. The identified pathogens exhibited varying levels of severity in branch canker and anthracnose on avocado branches and leaves. Furthermore, pathogenicity evaluations shed light on the potential of these pathogens to induce severe symptoms, emphasizing the urgency for effective control measures. The exploration of cultural and biological control strategies are crucial for mitigating the impact of branch canker, dieback, and anthracnose diseases, ensuring the sustainability of avocado cultivation in the region.

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 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.

Characterization and Pathogenicity of Botryosphaeriaceae Species Associated with Gummosis, Dieback, Trunk and Branch Cankers of Almond Trees in Türkiye

Members of Botryosphaeriaceae family with 25 genera and several species are spread over a wide range of lands and climates worldwide. They cause gummosis, decline, dieback and blight on many woody plants. The purpose of present study was to diagnose the pathogens linked to the aforementioned symptoms on almond trees in seven orchards of Yozgat province (Türkiye) with a DSb type climate (Hot humid continental - Köppen Geiger system of climatic classification).These trees indicated and displayed dieback, gummosis trunk and branch canker symptoms. They were identified by cultural and morphological characteristics, and compared by phylogenetic sequencing of the ITS regions, EF-1α and β-tubulin genes with those of other species in GenBank (NCBI). Diplodia seriata, Lasiodiplodia theobromae, Neofusicoccum parvum and Botryosphaeria dothidea were identified in 72 isolates based on the colony and conidial characteristics. Successful pathogenicity tests were carried out on two-year-old almond seedlings of cv: Ferredual using Koch’s postulates. The results validated the identification According to available literature on the subject, identification of B. dothidea was done for the first time on almond trees in Türkiye. Accurate identification, prevalence and incidence of the pathogens are crucial for developing effective disease management strategies to arrest disease outbreaks in Türkiye.

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 tiptransfer 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 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 β-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 Neofusicoccum parvum causing leaf target-spot disease on Ficus carica L. in China

First report of <i>Neofusicoccum parvum</i> causing leaf target-spot disease on <i>Ficus carica</i> L. in China

Draft genome sequences for four isolates of the hemp (Cannabis sativa) fungal pathogen Neofusicoccum parvum.

Four isolates of Neofusicoccum parvum, collected from diseased hemp (Cannabis sativa) plants over a period of 2 years and shown to be pathogenic on C. sativa, were examined in this study. Their genome sizes ranged between 42.8 and 44.4 Mb, with 16,499 ± 72 predicted genes across the four isolates.

The Comparative Genomics of Botryosphaeriaceae Suggests Gene Families of Botryosphaeria dothidea Related to Pathogenicity on Chinese Hickory Tree.

Trunk canker poses a major threat to the production of Chinese hickory tree (Carya cathayensis Sarg.), which is primarily determined by Botryosphaeriaceae. In our previous work, we identified Botryosphaeria dothidea as the predominant pathogen of this disease. However, it is still unclear about corresponding gene families and mechanisms associated with B. dothidea's pathogenicity on Chinese hickory tree. Here, we present a comparative analysis of high-quality genome assemblies of Botryosphaeria dothidea and other isolated pathogens, showing highly syntenic relationships between B. dothidea and its closely related species and the conservative evolution of the Botryosphaeriaceae family. Higher GC contents were found in the genomes of B. dothidea and three other isolated pathogens (Botryshaeria cortices, Botryshaeria fabicerciana, and Botryshaeria qingyuanensis) compared to Macrophomina phaseolina, Neofusicoccum parvum, Diplodia corticola, and Lasiodiplodia theobromae. An investigation of genes specific to or expanded in B. dothidea revealed that one secreted glucanase, one orsellinic acid biosynthesis enzyme, and two MFS transporters positively regulated B. dothidea's pathogenicity. We also observed an overrepresentation of viral integrase like gene and heterokaryon incompatibility proteins in the B. dothidea's genome. In addition, we observed one LRR-domain-containing protein and two Sec-domain-containing proteins (Sec_1 and Sec_7) that underwent positive selection. This study will help to understand B. dothidea's pathogenicity and potential influence on the infection of Chinese hickory, which will help in the development of disease control and ensure the security of Chinese hickory production.

Green synthesis of Superparamagnetic Iron Oxide and Silver Nanoparticles in Satureja hortensis Leave Extract: Evaluation of Antifungal Effects on Botryosphaeriaceae Species.

In recent years, green synthesis methods of metallic nanoparticles (MNPs) have been attractive because of the more facile, cheaper, and appropriate features associated with biomolecules in MNPs biosynthesis. This research represented an easy, fast, and environmentally friendly method to biosynthesis of superparamagnetic iron oxide nanoparticles (SPIONPs) and silver nanoparticles (AgNPs) by the Satureja hortensis leaf extract as stabilizer and reducer. The SPIONPs synthesized in co-precipitation method. The biosynthesized SPIONPs and AgNPs were studied their antifungal effects against three Botryosphaeriaceae plant pathogens, Botryosphaeria dothidea, Diplodia seriata, and Neofusicoccum parvum. UV-visible spectra (UV-Vis), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), field emission scanning electron microscopy (Fe-SEM), energy-dispersive X-ray spectroscopy (EDX), and vibrating-sample magnetometer (VSM) analyses were used to evaluate the physicochemical properties and verify the formation of green synthesized SPIONPs and AgNPs. UV-Vis spectra revealed absorption peaks at 243 and 448 nm for SPIONs and 436 nm for AgNPs, respectively. Microscopic and XRD analysis showed that SPIONPs and AgNPs was found spherical in shape with an average particle size of SPIONPs and AgNPs 10 and 12 nm, respectively. The antifungal test against Botryosphaeriaceae species showed that SPIONPs and AgNPs possessantifungal properties against B. dothidea, D. seriata, and N. parvum. However, AgNPs exhibits greaterantifungal activity than SPIONPs. Theresultsof thecytotoxicitytests of SPIONs and AgNPs on the MCF-7 cell line showed that AgNPs was significantly more cytotoxictowardsthe MCF-7 cell line, whereas nosignificant cytotoxiceffect was recorded by SPIONs. Therefore, these biosynthesized MNPs could be substituted for toxic fungicides that are extensively applied in agriculture and contribute to environmental health and food safety.

Modeling the Effect of Temperature on the Severity of Blueberry Stem Blight and Dieback with a Focus on Neofusicoccum parvum and Cultivar Susceptibility

Stem blight and dieback rank among the most relevant diseases affecting blueberry production worldwide. In Northern Italy, Neofusicoccum parvum, Diaporthe rudis, Cadophora luteo-olivacea and Peroneutypa scoparia have been reported to cause stem blight and dieback in blueberry. Considering that the incidence and severity of these diseases are on the rise in Northern Italy, two of the main aims of the present study were a—to compare the in vitro growth rate of the four fungi at different temperatures and b—to assess the aggressiveness of the same fungi on four commercial blueberry cultivars. Neofusicoccum parvum had the fastest growth rate and was the most aggressive pathogen. A possible effect of temperature on host colonization by N. parvum and disease expression was postulated and tested as a third aim. In planta trials were performed to model and predict the influence of temperature on the severity of blueberry stem blight and dieback caused by N. parvum. Increasing temperatures boosted the aggressiveness of the pathogen, causing higher disease severity and host mortality. Our findings suggest that temperature plays a relevant role in the severity of blueberry stem blight and dieback caused by N. parvum. Given the predictions of a warmer climate, this disease may become increasingly more significant and should be actively managed.

Postharvest diseases of mangoes in Fiji

ABSTRACT Despite considerable research globally on postharvest diseases of mango, virtually no work has been done to determine the occurrence of these diseases and their causal agents in Fiji. This study is the first major field and market survey of postharvest diseases of mango in Fiji. For the field survey, fruits from five local mango cultivars were harvested at each of five locations. Fruits from five imported cultivars were also harvested from one location in Nadi. For the market survey, vendors were selected at five municipal markets for fruit collection, along with ten roadside stalls. For field and market surveys, fruits were incubated at 23°C and assessed for postharvest disease (incidence and severity of body rots and stem end rots) when ripe. Isolations were made from disease lesions and fungal cultures were identified using multilocus sequence typing. High incidences of body and stem end rots were recorded across all surveys. In the field survey of local cultivars, ‘Salusalu’ fruits were found to have the lowest severity of body rot and stem end rot on average, while for imported cultivars, ‘Nam Doc Mai’ had the lowest average severity of these diseases. The market survey showed that ‘Salusalu’ had a lower incidence of anthracnose on the body of fruit compared to all other cultivars, although it did have a surprisingly high incidence of stem end rot at some localities. Fungal isolates from anthracnose lesions in the field and market surveys were identified as Colletotrichum asianum, C. simmondsii and C. fructicola. C. asianum was the predominant species associated with anthracnose symptoms, accounting for 97% of isolations. Lasiodiplodia theobromae and Neofusicoccum parvum were the predominant species found in association with mango stem end rot symptoms. Other species isolated from mango stem end rot in lesser numbers included L. brasiliensis, N. umdonicola and N. kwambonambiense. All of these fungi represent new reports for Fiji.