Stem-end rot (SER) causes brown necrotic lesions in the pulp near the base of the fruit pedicel and is one of the most devastating postharvest diseases of avocados in all avocado growing regions of the world. China\'s avocado industry is growing very rapidly, and the planting area is expanding, but little is known about the pathogens and genetic diversity of avocado SER. To determine the causal agents of SER, avocado fruits were sampled from the main avocado-producing areas in China during 2020 and 2021. Fungal isolates were obtained from SER symptomatic avocado fruits and identified by morphology combined with phylogenetic analysis of internal transcribed spacer (ITS), translation elongation factor 1-α (EF1-α) and β-tubulin (TUB2) gene sequences. All 101 isolates belonged to Lasiodiplodia spp., and four Lasiodiplodia species were identified, namely L. pseudotheobromae (59.41%), L. theobromae (24.75%), L. mahajangana (7.92%), L. euphorbiaceicola (1.98%), and six others are classified as Lasiodiplodia sp. (5.94%). There were only slight morphological differences in colonies and conidia of these four species of Lasiodiplodia. The pathogenicity tests showed symptoms of SER, and the 92.08% of the isolates exhibited a high level of virulence on avocado (disease index > 70), related to the disease severity on avocado fruit. All tested isolates grew well under conditions from 23 to 33℃. There was a significant difference in mycelial growth between the four species of Lasiodiplodia after treatment with high temperature or low temperature. L. pseudotheobromae growth was the fastest at 13 to 18℃, but was the lowest at 38℃ (P < 0.05). Red pigment could be produced by all tested isolates after culturing for 7 days at 38℃. The mycelial growth rate was the fastest on PDA medium, and the slowest on OMA medium but promoted spore formation (P < 0.05). In addition, was determined the genetic diversity of Lasiodiplodia pathogenic species associated with SER collected from avocado, mango, guava and soursop fruits was determined. A total of 74 isolates were clustered into 4 main ISSR groups by unweighted pair-group method with arithmetic mean (UPGMA) analysis, and the classification of this group was related to the host. Extensive diversity was detected in the Lasiodiplodia populations. The diverse geographical origins and host species significantly influenced the population differentiation, and most of the genetic variation occurred within populations (P < 0.001). This is the first study to identify the major pathogens of avocado SER in China and to survey their occurrence, pathogenicity and include a comparative analysis of genetic diversity with Lasiodiplodia spp. causing SER on other fruit hosts. Collectively, the Lasiodiplodia species complex affecting avocado showed high pathogenicity and diversity, while L. pseudotheobromae was the most frequently isolated species in China. The results of this study provide insights into the aspects of epidemic of SER disease caused by Lasiodiplodia species, which will help in developing strategies for the management and control of stem end-rot in avocado.

Stem-end rot disease has been causing damage to the production of pomelos in Vietnam. The cur-rent study aimed to (i) isolate fungal pathogens causing pomelo stem-end rot disease (PSERD) and (ii) discover Trichoderma spp. that had an antagonistic ability against pathogens under in vitro conditions. Fungi causing PSERD were isolated from pomelo fruits with symptoms of stem-end rot disease and collected from pomelo farms in Ben Tre province, Vietnam. Moreover, 50 fungal strains of Trichoderma spp. also originated from soils of these pomelo farms in Ben Tre province and were dual-tested with the fungal pathogen on the PDA medium. The results demonstrated that 11 pathogenic fungi causing PSERD were isolated from the fruit and showed mycelial growth of roughly 5.33-8.77 cm diameter at 72 h after inoculation. The two fungi that exhibited the fast-est growth, namely, S-P06 and S-P07, were selected. ITS sequencing of the S-P06 and S-P07 fungi resulted in Lasiodiplodia theobromae. All the 50 Trichoderma spp. strains were allowed to antago-nize against the S-P06 and S-P07 strains under in vitro conditions. The greatest antagonistic effi-ciency was found in Trichoderma spp. T-SP19 at 85.4-86.2% and T-SP32 at 84.7-85.4%. The two antagonists were identified as Trichoderma asperellum T-SP19 and T-SP32. The selected strains of Trichoderma asperellum were potent as a biological control for fruit plants.

Two of the major postharvest diseases impacting grapefruit shelf life and marketability in the state of Florida (USA) are stem-end rot (SER) caused by Lasiodiplodia theobromae and green mold (GM) caused by Penicillium digitatum. Here, we investigated the in vitro and in vivo efficacy of vapors of thymol, a natural compound found in the essential oil of various plants and the primary constituent of thyme (Thymus vulgaris) oil, as a potential solution for the management of GM and SER. Thymol vapors at concentrations lower than 10 mg L-1 significantly inhibited the mycelial growth of both pathogens, causing severe ultrastructural damage to P. digitatum conidia. In in vivo trials, the incidence and lesion area of GM and SER on inoculated grapefruit were significantly reduced after a 5 d exposure to 50 mg L-1 thymol vapors. In addition, the in vitro and in vivo sporulation of P. digitatum was suppressed by thymol. When applied in its vapor phase, thymol had no negative effect on the fruit, neither introducing perceivable off-flavor nor causing additional weight loss. Our findings support the pursuit of further studies on the use of thymol, recognized as safe for human health and the environment, as a promising strategy for grapefruit postharvest disease management.

Mango (Mangifera indica L.), belongs to the family Anacardiacea, and is one of the most popular tropical fruits in the world. Stem-end rot is a major postharvest disease of mango fruit, causing severe losses during storage in China (Chen et al., 2015). In July 2021, the mango fruits harvested from Baise Municipal National Agricultural Science and Technology Park (23.683568 N, 106.986325 E) of Guangxi province in China developed stem-end rot during storage. The disease incidence reached ca. 8.3%. The initial symptoms appeared as light brown lesions surrounding the peduncle, which quickly expanded becoming large dark-brown lesions. Small pieces of epidermis (5 mm × 5 mm) from 8 typical diseased friuts were cut from the edges of lesions surface-sterilized with 2% sodium hypochlorite and rinsed with sterile distilled water. The tissue was plated on potato dextrose agar (PDA) and incubated at 28 ℃ in the dark for 3 days. Fifteen, similarcolonies were isolated from the symptomatic tissue. The representative isolates DF-1, DF-2 and DF-3 were selected for morphological characterization, molecular identification, and pathogenicity testing. The colonies were circular with fluffy aerial mycelium, initially white turning to smoke-gray from the center in upper side and greenish black in reverse side, covering the 90 mm diameter Petri dish after 4 days of incubation on PDA at 28 ℃ in dark. Pycnidia were produced on the surface of the colony after 30 days. Conidia were fusiform, aseptate, hyaline, thin-walled with granular contents, apex sub-obtuse, base subtruncate to bluntly rounded, 14.0-20.3 (16.8±1.6) μm × 3.1-7.2 (5.1±0.9) μm (n=50). The sexual stage was absent. Based on morphology, isolates were preliminarily identified as Botryosphaeria speices. To accurately identify the pathogen, genomic DNA was extracted from the mycelium of the three isolates DF-1, DF-2 and DF-3. The internal transcribed spacer of rDNA region (ITS), elongation factor 1-alpha (EF-1α) and beta-tubulin gene (TUB) genes were amplified using primers ITS1/ITS4, EF1-728F/EF1-986R and Bt2a/Bt2b, respectively (Slippers et al., 2004). The nucleotide sequences were all deposited in GenBank (ITS: OP729176-OP729178 EF-1α: OP758194-OP758196 and TUB: OP758197-OP758199). Based on the BLASTn analysis, the ITS, EF1-α and TUB sequences of three isolates were 100%, 99% and 99% similar to the Botryosphaeria fabicerciana MFLUCC 10-0098 sequences (ITS: JX646789, EF-1α: JX646854 and TUB: JX646839). Multi-locus phylogenetic analyses (ITS, EF-1α and TUB) showed that the isolate DF-1, DF-2 and DF-3 were clustered within Botryosphaeria fabicerciana clade based on the maximum likelihood , Bayesian inference, and maximum parsimony methods. The pathogenicity test was performed by placing discs mycelium around the peduncle of mature mango fruits by pin-prick method. Each treatment carried out with 12 fruits. The inoculated fruits were placed in plastic boxes at 28 ℃ with three replicates. Three days after inoculation, typical symptoms of stem-end rot were observed. The control fruits were inoculated with sterile PDA discs, and remained symptomless. The same fungus was re-isolated from the symptomatic tissue to complete Koch\'s postulate. Botryosphaeria fabicerciana (basionym: Fusicoccum fabicercianum) was first reported as pathogen causing senescent twig of Eucalyptus spp. in China (Chen et al., 2011; Phillips et al., 2013). To our knowledge, this is the first report of Botryosphaeria fabicerciana causing stem-end rot of Mangifera indica in China.

Fungi of the family Botryosphaeriaceae are considered responsible for various symptoms in avocado such as dieback, external necrosis of branches and inflorescences, cankers on branches and trunks, or stem-end rot of fruits. In recent years, these problems are becoming more frequent in avocado orchards in the Canary Islands (Spain). This work includes the characterization of fungal species involved in these diseases, which were isolated from avocado crops in Tenerife Island between 2018 and 2022. A total of 158 vegetal samples were collected, from which 297 fungal isolates were culture-isolated. Fifty-two of them were selected according to their morphological features as representative isolates of Botryosphaeriaceae, and their molecular characterization was carried out, sequencing the ITS1-2 region as well as the β-tubulin and the elongation factor 1-alpha genes. Five species of Botryosphaeriaceae were isolated, including Neofusicoccum australe, N. cryptoaustrale/stellenboschiana, N. luteum, N. parvum, and Lasiodiplodia brasiliensis. This is the first time that L. brasiliensis has been associated with avocado dieback and that N. cryptoaustrale/stellenboschiana has been cited in avocado causing symptoms of dieback and stem-end rot. However, it was not possible to assign our isolates unequivocally to N. cryptoaustrale or N. stellenboschiana even additionally using the rpb2 marker for their molecular characterization. Botryosphaeriaceae family seem to be involved in avocado dieback, in the premature fall of fruits during their development in the field and in post-harvest damage in Tenerife, but further studies are needed to clarify the fungal pathogens associated with symptoms in relation to phenological plant growth stages or less frequently observed.

This study evaluated the antifungal activity of ozone (O3) against stem-end rot of mango fruit (cv. Keitt). Mango fruit were exposed to gaseous ozone (0.25 mg/L) for 24 or 36 h during cold storage, and control fruit were untreated. Experimental fruit were stored at 90% relative humidity and 10 ± 0.5 °C for three weeks and ripened at ambient temperature for one week. Ozone treatment (24 h) inhibited the mycelial growth of Lasiodiplodia theobromae by 60.35%. At day twenty-eight of storage, fruit treated with O3 for 36 h had low mass loss (%) and high firmness compared to the untreated control fruit. Treating mango fruit with O3 (36 h) maintained the color and concentration of total flavonoids throughout the storage time. At the end of storage, peroxidase activity under the O3 24 h treatment was significantly higher (0.91 U min-1 g-1 DM) compared to O3 (36 h) and control, which, respectively, had 0.80 U min-1 g-1 DM and 0.78 U min-1 g-1 DM. Gaseous ozone for 24 h is recommended as a cost-effective treatment for controlling stem-end rot. These findings suggest that gaseous ozone effectively controlled stem-end rot and enhanced the postharvest quality of mango fruit.

Mango (Mangifera indica L.) is considered one of the most important tropical or subtropical fruit crops (Nelson et al.2008). China is the second-largest producer of mango (Kuhn et al. 2017). In June 2021, postharvest stem-end rot disease was observed on Narcissus mango (about 20% of the fruits showed similar symptoms of infections) in local agricultural market of Guangzhou, China. Black rot symptomatic lesions were observed on the fruit surface, which initially started from the stem end and progresses into decay, turning brown. To isolate and identify the pathogen, small pieces (3-5 mm2) were excised from the lesion margins of the fruits (n=54), which were surface sterilized by 1% NaOCl (1 min), 70% ethanol (30 s) and then washed twice with sterile distilled water. After sterilization, the tissues were cultured on potato dextrose agar (PDA). Three morphologically similar isolates (SXM-1/2/3) were obtained and the representative isolate SXM-1 was analyzed. Colonies surface initially had white-gray moderate aerial mycelia, in reverse umber with patches of pale luteous to luteous. On malt extract agar (MEA) surface dirty white, reverse greyish sepia with patches of sienna. Conidiomata pycnidia, black, erumpent to superficial on PDA, globose with neck, ostiole exuding cream conidial droplets; Conidiophores hyaline, smooth, 1-3-septate, branched, densely aggregated, cylindrical, straight to sinuous, 22-42 × 2.8-3.7 µm. Alpha conidia (n = 50) aseptate, hyaline, smooth, fusiform to somewhat short cylindrical, 3.2-11 ×1.3-3.5 µm. Beta conidia (n = 30) hyaline, smooth, curved or hamate 14.8-33.6 × 1.1-2.6 μm. According to morphological characterization, the representative isolate SXM-1 was similar to Diaporthe pseudomangiferae CBS 101339 (Gomes et al. 2013). For molecular identification, the internal transcribed spacer (ITS) region, histone H3 (HIS) and β-tubulin (TUB) genes (White et al. 1990; Carbone et al. 1999; Glass et al.1995) were amplified and sequenced, which were deposited in GenBank (ON243823, ON254656, ON254655). BLASTN analysis revealed that DNA sequences of the isolates (SXM-1/2/3) showed 99% identity with those of D. pseudomangiferae (MG576128.1, KC344149, MN329124.1), respectively. A phylogenetic tree analysis based on the concatenated sequences confirmed the isolates as D. pseudomangiferae. Pathogenicity tests were made with the representative isolate SXM-1. Healthy fruits were inoculated with 5 mm mycelial discs of the representative isolate SXM-1 after being wounded with a needle or non-wounded, control fruits were inoculated with sterilized PDA plugs. All inoculated and control fruits were incubated in the dark at 26°C for 7 days post-inoculation. Control fruits remained asymptomatic, whereas inoculated fruits were dark brown necrotic lesions with a roughly circular shape around the inoculation sites. Pathogenicity tests were performed in triplicate. The pathogenic isolates were successfully reisolated, thus confirming Koch\'s postulates. D. pseudomangiferae was associated with fruit peel of mango in Mexico and the Dominican Republic, and it has also been reported to cause inflorescence rot, rachis canker, and flower abortion in mango (Gomes et al. 2013; Serratodiaz et al. 2014). To our knowledge, this is the first report of D. pseudomangiferae causing postharvest stem-end rot of mango fruits in China. This finding suggests that D. pseudomangiferae is a potential problem for mango fruit production in China, and it is important to establish an adequate and effective control management of this disease. References: Nelson, S. C. 2008.Mango Anthracnose (Colletotrichum gloeosporiodes). Publication PD-48. Cooperative Extension Service, College of Tropical Agriculture and Human Resources, University of Hawai\'i at Manoa, U.S.A. Kuhn, D. N., et al. 2017. Front. Plant Sci. 8:577. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego. Carbone, I., et al. 1999. Mycologia. 91:553. Glass, N. L., et al. 1995. Appl. Environ. Gomes R. R., et al. 2013. Persoonia. 1:31. Serratodiaz, L. M., et al. 2014. Plant Dis. 98:1004. * These authors contributed equally to this work and should be considered co-first authors. The authors declare no conflict of interest. Keywords: Stem-end rot, Diaporthe pseudomangiferae, Mango, China.

Tobacco (Nicotiana tabacum L.), is a major cash crop grown worldwide for its leaves, which are dried and fermented before being put in tobacco products. Tobacco production is seriously threatened by numerous diseases (Qiu et al. 2021). In August 2021, plants with stem-end rot were observed in a tobacco field in Zunyi City, Guizhou Province, China. Surveys indicated a 22 to 35% disease incidence in five counties. Symptoms of black necrosis appeared at the base of stems, and leaves turned yellow. To isolate the pathogen, diseased stems were cut into small segments and placed on potato dextrose agar (PDA) at 25°C in darkness for 3 to 5 days. To obtain pure cultures, hyphal tips from colonies were transferred to fresh PDA plates. A representative strain, GZAX 110, was used for further identification. The fungal colonies were initially gray, later deepening to smoke-gray. Conidiogenous cells were fully divided, discrete, transparent, thin-walled, smooth and cylindrical. Conidia matured slowly, were ellipsoid to ovoid, containing granular content, with a rounded apex. The base was largely truncated, and conidia became dark brown with one central septum, 21.0-30.0 × 12.0-18.0 μm. On water-agar medium, teleomorph structures were not observed. These characteristics suggested the fungus was Lasiodiplodia sp. (Netto et al. 2014). For further confirmation, genomic DNA was extracted using the CTAB method (Watanabe et al. 2010); and the ITS (internal transcribed spacer region of rDNA) and EF-1α (translation-elongation factor) regions were amplified with primer pairs ITS1/ITS4 and EF1-688F/EF1-1251R (Cruywagen et al. 2017), respectively. The ITS and EF-1α sequences (OM534558 and OM632673) were analyzed by BLASTN searches. The ITS sequence showed 100% identity (490/490 bp) to L. brasiliense strain AGQMy0011 (MW274145) and the EF-1ɑ sequence showed 100% identity (551/551 bp) to L. brasiliense strain EX1 (MF580811). A multilocus phylogenetic tree was constructed via the Maximum-likelihood (RAxML v.7.2.8) and Bayesian Inference (MrBayes v.3.2.1) analyses (Elsie et al. 2017) using combined ITS and EF1-α reference sequences of Lasiodiplodia species. Phylogenetic analysis showed that GZAX 110 clustered monophyletically with strains of L. brasiliense. Thus, the isolate GZAX 110 was confirmed as L. brasiliense. Pathogenicity of GZAX 110 was tested on tobacco plants at the eight leaf stage by attaching mycelial plugs (5 mm in diameter) to stems and leaves according to Cruywagen et al. (2017). Inoculated plants were kept in a greenhouse (16 h light/8 h darkness, 22℃, relative humidity >85%). Control plants were inoculated with PDA plugs. The experiment was repeated three times with five plants. Seven days after inoculation, dark brown necrosis was observed at inoculation sites on stems and leaves, while the control plants remained healthy. The pathogen was re-isolated from the inoculated sites and further validated as the same fungus through morphological and phylogenetic analyses. Previously, this fungus has been reported on Mangifera indica (mango) in China (Zhang et al. 2018), and apple (Martins et al. 2018) and papaya (Netto et al. 2014) in Brazil. However, to our knowledge, this is the first worldwide report of L. brasiliense causing stem-end rot on tobacco. This report provides information for future diagnosis and management of the disease.

US banana producers are looking for the organic banana market in the southeastern US including Florida and the coastal region of Georgia (Schupska, 2008). In December of 2020, a 6-hand bunched banana (cv. Pisang Awak, belongs to tetraploid AABB genome) with nearly 50% infection (with 15-20% disease severity, <1% of the total harvest) was received from the UGA Banana Research Plot, Tifton, GA with typical stem end rot symptoms of softened and water-soaked flesh. To identify the pathogen, the infected tissues were separated with a sterilized blade, surface disinfested with 10% bleach solution for 1 min, and subsequently washed in three changes of sterile distilled water. The sterilized tissues were aseptically placed on potato dextrose agar (PDA) medium and incubated at 25°C in the dark for 5-10 days. Two isolates of the pathogen with similar colony morphology were obtained and initially identified morphologically using a Botryosphaeriaceae taxonomic key (Phillips et al., 2013). The first growth phase for the isolates documented on PDA, gave rise to white colonies, followed by a dense, black mycelium. The mycelium was fast-spreading, immersed, branched, and septate. The shiny black pycnidia were viewed on the PDA surface after 8-10 days of incubation. Initially, the morphological features of the isolates were identified as Lasiodiplodia spp. (Phillips et al., 2013). To identify to species level, genomic DNA was extracted from two isolates (SW1 & SW2) and amplified by PCR for sequencing using ITS1/ITS4 (White et al., 1990), EF1- 688F/ EF1- 1251R (Alves et al., 2008), Bt2a/Bt2b (Glass & Donaldson, 1995) and rpb2-LasF/ rpb2-LasR (Cruywagen et al., 2017). The ITS (MZ293097 and MZ293114), EF1(OL657173 and OL657174) and rpb2 (OL704860 and OL704861) sequences showed 100% identity and Bt (OL657175 and OL657176) sequences showed 99.5% and 99.7% identity to the corresponding sequences of Lasiodiplodia brasiliensis type strain CMW35884 in GenBank (ITS: KU887094, EF1: KU886972, Bt: KU887466 and rpb2: KU696345). To further affirm the identity, a concatenated phylogenetic analysis was executed with ITS, EF1, Bt, and rpb2 sequences of both isolates and 31 reference strains using Geneious Prime 2019.2.3 Tamura-Nei Neighbor-joining method with 1,000 bootstrap replications, and the outcome was consistent with the conclusion above. To fulfill Koch\'s postulates, a pathogenicity test was performed with bunched bananas. Two whole bunched bananas surface sterilized with 10% bleach solutions and subsequent washing with sterilized water were cut into 3 bananas per brunch. The inoculum was prepared with 105 spores/ml. The conidial suspension was inoculated on the on-cut surface of the banana crown (300 µl per crown) using a micropipette. Sterile distilled water was applied as a control. The fruit was then packed and sealed in plastic bags and incubated at 25°C. Stem end rot symptoms were first appeared at 5 dpi and increased 7 days later. Two weeks post-inoculation, typical blackened and softened rot tissues were observed, and control fruits remained asymptomatic. To the best of our knowledge, this is the first report of Lasiodiplodia brasiliensis causing stem-end rot of bananas in the USA. This report would be valuable to the banana growers in the southeastern US by taking suitable control measures to confront this fungal disease.

The focus of this study was to develop technologies using chlorine dioxide (ClO2) gas to control postharvest stem-end rot of citrus caused by Lasiodiplodia theobromae. Mycelial growth of L. theobromae on potato dextrose agar (PDA) plugs was completely inhibited by a 24-h ClO2 exposure provided by 0.5 g of solid ClO2 generating granular mixture in a 7.7-liter sealed container. In vivo experiments were conducted on artificially inoculated Tango and naturally infected U.S. Early Pride mandarins. When ClO2 treatments were initiated 0 to 6 h after inoculation, decay development was significantly reduced as compared with the control, and higher ClO2 doses were more effective. A ClO2 treatment (using 3 g of generating mixture per 7.7-liter sealed container) administered 0 h after inoculation resulted in 17.6% Diplodia stem-end rot incidence compared with 95.6% in the control, whereas the same treatment administered 24 h after inoculation was much less effective, resulting in 63.0% incidence compared with 85.4% in the control. Diplodia stem-end rot incidence of naturally infected fruit after using 6 or 9 g of generating mixture per 24-liter sealed box was 23.8 or 25.7%, respectively, compared with 47.9% for control fruit. The ClO2 treatments had no negative effects on fruit quality characteristics including weight loss, firmness, puncture resistance, titratable acids (TAs), total soluble solids (TSSs), and rind color. Albedo pH at wounds was significantly reduced from 6.0 to 4.8 by the ClO2 treatments, whereas undamaged albedo remained at 5.8. In addition, no visible physiologic defects, such as peel browning and bleaching, were observed on ClO2-treated fruit. These results indicate that ClO2 gas has the potential to be developed as a component of an integrated citrus postharvest decay control system to minimize fruit losses.