Fruit Rot Research Articles

Exploration of Pathogenic Microbes and Antagonistic Agents in Apple Production Centers in Malang Regency

The apple farming group is one of the built of Department of Plant Pests and Diseases, Agricultural Faculty, Brawijaya University. In 2019, this departmen received a request from apple farmers in Madiredo Village, Pujon District to held an assistance programs of apple cultivation and environmentally pest control. This is related with the increasing of fruit rot and other diseases which is caused a decrease of apple production especially in Malang Regency. In general, apple farmers still rely on disease control using chemical fungicides which can have a negative impact on the environment and create resistance to pathogenic fungi. Therefore, this research was carried out to explore and study the potential of biological agents in apple production centers in Malang Regency, specifically in Poncokusumo, Pujon, and Batu District on conventional fields and mixed fields. This research was held observationally through fields surveys and farmer interviews, took soil samples and disease samples then then isolating, identifying and testing for antagonism at laboratory. Based on interviews and observation of symptomology, two dominant diseases were found, namely fruit rot disease (Gloeosporium sp.) and marsonina leaf spot disease (Marssonina coronaria). The results of exploration of the rhizospheric apple plants obtained isolates on conventional fields in Kec. Pujon has the highest abundance of fungal colonies compared to other regions. Based on the results of the antagonism test for pathogenic fungi and biological agents, it was found that the isolate codes KH.C, KK.A, JK.B, JK.J, and BK.B had the highest percentage of inhibition or the most effective in controlling the pathogen Gloeosporium sp. So in the next, the species can be identified molecularly.

First Report of the Occurrence of Diaporthe nobilis Causing Postharvest Fruit Rot on ‘Shine Muscat’ Grape in Yuncheng, China

In recent years, ‘Shine Muscat’ (Vitis labruscana × Vitis vinifera) has been the most popular table grape among consumers in Asian countries (Choi et al., 2021). In November 2020, fruit rot was observed in packing house 2 months after cold-stored in Yuncheng (35°16′N, 111°08′E), Shanxi Province, China. Of 38 boxes (5 kg per box) grape were selected randomly from the packing house with a stock of 7,000 kg. The incidence of grape rot was 71.5%, observed in 143 of the 200 clusters sampled. The diseased fruit showed symptoms including pedicel browning, and extending lesions and softening of berries. Samples (1 mm2) cut from the border between diseased and healthy tissue were surface sterilized with 70% ethanol for 1 min, then rinsed with sterile distilled water thrice, and placed on potato dextrose agar (PDA) plate 25 ° C in the dark for 5 days. After subculturing for three times, 26 isolates with a similar type of fungal colony were obtained. Colonies on PDA was initially white fluffy aerial, but gradually turned grey. Pycnidia were black and globes, producing α and β conidia. α conidia abundant, aseptate, hyaline, ellipsoid, both ends obtuse, biguttulate, 6.52~9.05 μm×2.19~3.25 μm (n=50). β conidia abundant, hyaline, aseptate, filiform, flexuous to J-shaped, 22.79~36.75 μm×1.24~1.92 μm (n=50). For further identification, phylogenetic analysis of combined internal transcribed spacer (ITS), β-tubulin (TUB), translation elongation factor (EF1-α) and calmodulin (CAL) datasets were carried out. The representative isolate SS1 and SS2 were selected for identification. Primers ITS1 and ITS4 (White et al. 1990) were used to amplify the ITS region. The gene fragments of TUB, CAL and EF1-α were amplified by the primers Bt2a/Bt2b (Glass and Donaldson 1995), CAL228F/CAL737R and EF1-728F/EF1-986R, respectively (Carbone and Kohn 1999). The sequences were deposited in GenBank (SS1: MW644526 for ITS, OQ718912 - 4 for CAL, TEF-1α, and TUB, respectively; SS2: MW644527 for ITS, OQ718915 -7 for CAL, TEF-1α, and TUB, respectively). BLASTn analysis of the NCBI database indicated that the ITS, TUB, EF1-α, and CAL sequences had very high nucleotide homology (98 to 100%) to ex-type sequences for D. nobilis (CBS 116953, Gomes et al., 2013) in GenBank (ITS: KC343147, TUB: KC344115, EF1-α: KC343873, and CAL: KC343389). Phylogenetic tree based on the combined sequences revealed that the two isolates clustered well with D. nobilis. To confirm its pathogenicity, 30 fresh-harvested ‘Shine Muscat’ grape fruits were used to mock inoculate. Wounds (5 mm wide by 2 mm deep) were created uniformly at the equator of the grape fruit with a sterile puncher and inoculated with mycelial plugs (5 mm in diameter) prepared from 7-day-old fungal cultures grown on PDA (Wild 1994). The control fruits were inoculated with agar disks without mycelium. Inoculated fruits were placed in a chamber with the 80% relative humidity at 25°C for 8 days. After inoculation, all treated fruits turned brown, with significant necrotic lesions; fruits in control showed no symptoms. Fungal colonies reisolated from inoculated fruits had similar morphological characteristics as D. nobilis. Diaporthe spp. are responsible for diseases on a wide range of plants hosts, causing root and fruit rots, dieback, cankers, leaf spots, blights, decay and wilt (Gomes et al., 2013). D. nobilis causing postharvest grape rot can seriously restrict the development of local grape industry.

A simple protocol for cultivating the bacterivorous soil nematode Caenorhabditis elegans in its natural ecology in the laboratory.

The complex interplay between an animal and its surrounding environment requires constant attentive observation in natural settings. Moreover, how ecological interactions are affected by an animal's genes is difficult to ascertain outside the laboratory. Genetic studies with the bacterivorous nematode Caenorhabditis elegans have elucidated numerous relationships between genes and functions, such as physiology, behaviors, and lifespan. However, these studies use standard laboratory culture that does not reflect C. elegans true ecology. C. elegans is found growing in nature and reproduced in large numbers in soils enriched with rotting fruit or vegetation, a source of abundant and diverse microbes that nourish the thriving populations of nematodes. We developed a simple mesocosm we call soil-fruit-natural-habitat that simulates the natural ecology of C. elegans in the laboratory. Apples were placed on autoclaved potted soils, and after a soil microbial solution was added, the mesocosm was subjected to day-night, temperature, and humidity cycling inside a growth chamber. After a period of apple-rotting, C elegans were added, and the growing worm population was observed. We determined optimal conditions for the growth of C. elegans and then performed an ecological succession experiment observing worm populations every few days. Our data showed that the mesocosm allows abundant growth and reproduction of C. elegans that resembles populations of the nematode found in rotting fruit in nature. Overall, our study presents a simple protocol that allows the cultivation of C. elegans in a natural habitat in the laboratory for a broad group of scientists to study various aspects of animal and microbial ecology.

First report of anthracnose fruit rot of papaya caused by Colletotrichum gigasporum in China.

Papaya (Carica papaya L.) belonging to the family Caricaceae is well known for its economic and nutritional value. Anthraconse caused by Colletotrichum spp. is a main postharvest disease of papaya fruit during storage (Cia et al., 2007). In July 2022, papaya fruits with anthracnose symptoms were collected in Changjiang County (108.996180E, 19.246560N), Hainan Province, China. The disease incidence of fruit rot reached 6.3%. Initial symptoms appeared as the watery lesions with tiny black spots, turning to dark brown, sunken necrotic lesions. The diseased tissues were cut into 18 pieces (5×5 mm) from 6 papaya fruits, disinfected with 2% sodium hypochlorite for 60 s, and rinsed three times with sterilized water. The pieces were air-dried and then placed on potato dextrose agar (PDA) at 28 ℃ for five days. Twelve isolates with similar morphology were obtained from 18 tissue pieces. Three isolates (FMG01, FMG02 and FMG03) were selected for morphological identification, molecular identification, and pathogenicity tests. Colonies were initially white, then gradually became dark grey on PDA. The ascospores were hyaline, fusoid, rounded at both ends, 37.43-84.32 (55.79±7.61) μm × 4.30-6.55 (5.36±0.60) μm (n=50). The conidia were hyaline, unicellular, long cylindrical, bluntly rounded at both ends, 11.59-25.54 (18.62±2.33) μm × 5.12-8.44 (7.19±0.62) μm (n=100). Appressoria were gray to dark brown, irregular, pyriform, or ovoid, 10.14-21.40 (13.81±2.25) μm × 6.05-11.85 (9.16±1.29) μm (n=50). Morphological features are similar to Colletotrichum gigasporum identified and described by Rakotoniriana et al (Rakotoniriana et al., 2013). In order to accurately identify the isolates, the internal transcribed spacer region (ITS) of the rDNA, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the partial actin (ACT), the beta-tubulin (TUB2) and the calmodulin (CAL) genes were amplified and sequenced (Cannon et al., 2012). The nucleotide sequences were deposited into GenBank (accession numbers, ITS: OR017446 to OR017448, GAPDH: OR042810 to OR042812, ACT: OR042813 to OR042815, TUB2: OR042816 to OR042818, CAL: OR042819 to OR042821). Based on the BLASTn analysis, these sequences were more than 99% with the reference strain CBS 125476 of C. gigasporum sequences (ITS: MH863698, GAPDH: KF687833, ACT: KF687790, TUB2: KF687875, CAL: KF687814), respectively. The results of the multilocal phylogenetic analysis showed that the three isolates were C. gigasporum based on the Maximum Likelihood and Bayesian inference method. The pathogenicity test was performed by wounded with a sterile needle on the surface-sterilized papaya fruits. The mycelial discs (5 mm diameter) of three isolates were inoculated orderly on the same fruit, and the same inoculation was applied to non-wound papaya fruits. The control group were inoculated with sterilized PDA. Each treatment carried out with 9 fruits. The inoculated fruits were placed at 28 ℃ in plastic preservation boxes (32×22×11 cm) with sterilized distilled water to maintain high humidity. After 5 d of inoculation, typical anthracnose symptoms were observed on wound fruits and the non-wound fruits developed symptoms at 7 dpi, control fruits were symptomless. The fungi re-isolated from the inoculated fruits lesions after inoculation and identified by morphological characterization and molecular identification, fulfilling Koch's postulates. C. gigasporum has been reported causing leaf rot of Dalbergia odorifera in China (Wan et al., 2018). To our knowledge, this is the first report of anthracnose fruit rot of papaya caused by C. gigasporum in China.

First report of Irpex rosettiformis causing white root rot in avocado trees in Michoacán, México.

In México, avocado production is an important economic source. In the last season it generated $ 3. 27 billion USD of foreign currency in the country. Irpex spp. are wood decay fungi. In the period 2019-2022, in the state of Michoacán (19°13' N; 101°55' W), México, basidiomes of Irpex sp. were observed on the base of trunks and crowns of 5-years-old and older avocado (Persea americana) trees. The trees exhibited disease symptoms that included white root rot, leaf yellowing, small leaves, branch diebacks, generalized defoliation, apical flaccidity, abundant but small sun burnt fruits due to the lack of foliage, and after 2-4 years of first disease appearance, the infected trees died. In the place where fungus was established, abundant white and cottony mycelium was formed, which caused trees decay. The incidence of the disease in the sampled orchards was estimated to be 30% per ha with 350 - 400 trees, which was determined through a simple sampling design focused on trees with signs and symptoms of the disease due to the phytopathogen. Samples of infected tissue (roots and stems) and fungal basidiomes were collected from 90 trees (5-6 per orchard). The symptomatic avocado trees studied were randomly selected from 17 orchards. For the fungal macroscopic characterization, the synoptic keys described by Gilbertson and Ryvarden (1986) and by Largent (1973) were used. The samples showed typical structures corresponding to Irpex sp., including rosettes, annual basidiomes, a system of monomitic hyphae, and subglobose basidiospores. In vitro fungal isolation from basidiomes and infected tree tissues was done according to the protocol of Agrios (2004). The fungal strains were maintained on PDA at 28 °C. At 16 days of incubation the colonies were opaque, whitish with fluffy and corky mycelium. Microscopic analysis of the fungus showed typical yellowish spores, with an ellipsoid shape of 3-4 x 4-5.5 µm (50 accounted structures per isolate [N=19]) and basidia of 20-25 x 4.5-5.5 µm (n=20 basidiomes). For molecular characterization, two molecular markers were used, the internal transcribed spacer rDNA-ITS1 5.8 rDNA-ITS2 (ITS; White et al. 1990) and the large ribosomal subunit (LSU; Vilgalys and Hester 1990). The PCR reaction was performed as described by Martínez-González et al. (2017). The consensus sequences were compared with those deposited in the NCBI-GenBank, using the BLASTN 2.2.19 tool (Zhang et al. 2000), the samples showed 99% match with the species, Irpex rosettiformis. GenBank accession numbers of the submitted isolates are summarized in supplementary Table 4. To test Koch's postulates, 3-months old avocado plants grown in greenhouse conditions were inoculated (n = 10 per each isolate [N= 19]) on the roots with 3 g of I. rosettiformis mycelium. The experiment was done twice with 20 non-inoculated plants as control. After 67 days, basidiomes (50 x 70 x 1.5 mm in average) were observed where the disease incidence was >77%, with subsequent tree decline. The pathogen was re-isolated in vitro in PDA and its identity was confirmed by morphological characteristics of mycelium. This work shows that I. rosettiformis is not only a wood decay fungus, but also a phytopathogen, the causative agent of white root rot disease in P. americana var. drymifolia, cultivar 'Hass', which establishes a precedent for monitoring and preventing its proliferation to other regions in the American continent and the world where nursery avocado seedlings are exported.

Biocontrol of Fruit Discoloring and Fruit Rot Disease in Longan (Dimocarpus longan) by Using Antagonistic Bacillus spp.

Fruit discoloration and fruit rot disease in longan is a serious disease caused by the pathogenic fungi Pestalotiopsis oxyanthi and Lasiodiplodia pseudotheobromae. This study investigated the potential of antagonistic bacteria as an eco-friendly alternative to traditional fungicides for managing longan diseases caused by these fungal pathogens. Two bacterial isolates, UP-JLS050 and UP-JLS067, demonstrated significant inhibitory activity against the pathogenic fungi P. oxyanthi and L. pseudotheobromae, with high inhibitory activity. The bacterial strains were isolated from leaves and soil in a longan garden, and dual culture methods showed that the isolates UP-JLS050 and UP-JLS067 exhibited the highest inhibitory activity on the fungal pathogens, with percentage inhibitions of 72.67 ± 2.31 and 70.37 ± 1.28 %, respectively. Biochemical methods and molecular techniques, including sequencing of the 16S rDNA gene, were then used to identify the isolates. This confirmed their similarity to Bacillus subtilis and Bacillus amyloliquefaciens with 100 and 99 % identity, respectively. In vitro tests on longan fruit indicated that these antagonistic bacteria, administered at a concentration of 108 CFU/mL, significantly reduced disease incidence by up to 45 % after 9 days of incubation. The incubation of B. subtilis (UP-JLS050) and B. amyloliquefaciens (UP-JLS050) reduced disease incidence by 45 ± 5.77 and 32.5 ± 5.00 %, respectively. HIGHLIGHTS Fruit Discoloration and Rot in Longan: Caused by pathogenic fungi, namely Pestalotiopsis oxyanthi and Lasiodiplodia pseudotheobromae Antagonistic Bacteria for Disease Control: This study explores the potential of antagonistic bacteria as an eco-friendly alternative to traditional fungicides for managing longan diseases caused by these fungal pathogens Effective Inhibition: Two bacterial isolates, UP-JLS050 and UP-JLS067, demonstrated significant inhibitory activity against oxyanthi and L. pseudotheobromae, with inhibitions exceeding 70 % on PDA medium Identification of Bacterial Strains: Molecular techniques, including 16S rDNA gene sequencing, confirmed the identity of these isolates as subtilis and B. amyloliquefaciens Reduced Disease Incidence: In vitro tests on longan fruit indicated that these antagonistic bacteria, administered at a concentration of 108 CFU/ml, significantly reduced disease incidence by up to 45 % after 9 days of incubation GRAPHICAL ABSTRACT

First Report of Fusarium luffae Causing Leaf Blight on Loquat in Eastern China.

During May-June 2021 and 2022, leaf blight symptoms were observed on loquat leaves (Eriobotrya japonica cv. 'Mogi') in Jiangsu Province (Xuzhou municipality, 117.17° E, 34.13° N) in China. Approximately 10% of the leaves on the two hundred trees studied in a six-year-old loquat orchard exhibited round lesions that changed from light yellow to reddish-brown in 8-10 days. Approximately 3% of the infected leaves exhibited numerous lesions that coalesced, leading to expansive blighted areas. Twenty-five samples of symptomatic tissue, approximately 0.2 cm2 in size, were collected in May 2022 from five different trees (five samples per tree), sterilized in 2% NaOCl for 1 min, washed twice with sterilized ddH2O, and incubated at 26°C for 5 days on PDA medium containing 50 µg/mL chloramphenicol. Six isolates were obtained via single spore isolation. ITS (OQ954852-OQ954857), TUB2 (OQ968488-OQ968493), EF1-α (OQ971890-OQ971895), RPB1 (OQ971896-OQ971901), and RPB2 (OR037266-OR037271) genes were amplified using the ITS1/ITS4, T1/T22, EF1-728F/EF1-986R, RPB1-R8/RPB1-F5, and fRPB2-7CF/fRPB2-11aR primers, respectively (O'Donnell et al. 2010). The species was identified using the Fusarioid ID database (Crous et al. 2021), revealing that all obtained isolates showed high homology to representative F. luffae strains. Upon combining the ITS, TUB2, EF1-α, RPB1, and RPB2 sequences, the isolates showed 99.42%-97.85% and 99.59%-98.10% identity to F. luffae CGMCC 3.19497 (ex-type strain) and NRRL 32522, respectively. A molecular phylogenetic tree was constructed using MEGA X, with a selection of representative Fusarium strains. Microscope observations showed septate mycelium, microconidia (6.86 ± 0.91 µm length, 1.67 ± 0.24 µm width, containing 1 septum; number of observations = 21), fusiform macroconidia (15.88 ± 1.43 µm length, 1.66 ± 0.24 µm width, containing 1 septum; number of observations = 45), and linear chlamydospores (79.36 ± 28.36 µm length, 12.03 ± 3.37 µm width; number of observations = 152). These observations are consistent with the morphology of F. luffae (Wang et al. 2019). All isolates exhibited identical morphological characteristics. All isolates were evaluated for pathogenicity in vivo using healthy non-detached loquat leaves. A total of 15 leaves from 5 different three-month-old 'Mogi' loquat trees were used for each isolate. Experiments were performed three times. A suspension of 1 × 106 spores/mL obtained from a seven-day-old colony (10 mL per 15 leaves), was sprayed on non-wounded leaves for inoculation. Sterilized ddH2O was used in the control experiment. Inoculated trees were stored at 26°C and 70% relative humidity for four days. Leaf blight symptoms were observed in all inoculated leaves, and the symptoms were observed in all repeated trials. The pathogen was recovered, and its identity was confirmed by ITS sequencing and morphological analysis, fulfilling Koch's postulates. In recent years, F. luffae has been reported to cause fruit rot on muskmelon, flower rot on kiwifruit, soybean pod rot, and leaf spot on cherry in China (Yu et al. 2022; Zhang et al. 2022; Zhao et al. 2022; Zhou et al. 2022), demonstrating the host promiscuity of this pathogen. Although F. solani has been identified as the causal agent of root rot and fruit rot on loquat (Abbas et al. 2017; Wu et al. 2021), this is the first report of F. luffae causing leaf blight on loquat worldwide. This report will help to understand the pathogens affecting loquat orchards in China.

First Report of Fruit Rot Caused by Fusarium luffae in Cherry Tomato in China.

Tomato (Solanum lycopersicum L.) is a fruit of great economic value that is grown worldwide. In November 2022, fruit rot symptoms were observed in cherry tomatoes (cv. Qianxi) in Jinan City of Shandong Province, China. Six cherry tomato samples (four symptomatic and two asymptomatic) were collected from commercial fields (approximately 1.2 ha) where the incidence of the disease ranged from 5 to 10%. The core and surface of the infected fruit were colonized and covered with white mycelia. Tissue pieces (5 mm × 5 mm) from the junction of healthy and diseased samples were surface-disinfected with 75% ethanol for 3 min, followed by 10% sodium hypochlorite for 5 min, and washed three times with sterile water. Tissue pieces were cultured on potato dextrose agar (PDA containing 200 mg/L timentin) at 28°C for five days. Four fungal isolates with similar morphological characteristics were obtained from each sample. Two representative isolates were collected and purified using the single-spore method. After five days on PDA at 28°C, FL1 and FL2 colonies showed abundant white to cream colored aerial mycelia with an average growth rate of 5 mm/day. On carnation leaf agar, FL1 was characterized by falcate macroconidia with pronounced dorsiventral curvature containing three to eight tapered apical cells and foot-shaped basal cells ranging in size from 25 to 74 μm × 3.6 to 6.8 μm (n=50). Microconidia and chlamydospores were not observed. These morphological characteristics were consistent with the description of F. luffae (Wang et al. 2019). DNA was extracted using the CTAB method. The nucleotide sequences of the translation elongation factor 1-α gene (TEF1) and the second largest RNA polymerase II subunit (RPB2) were amplified using specific primers EF1/EF2 and RPB2F/R, respectively (O'Donnell et al. 1998, 2010). FL1 and FL2 sequences were deposited in GenBank (TEF1: OQ427345 and OQ427346, RPB2: OQ427347 and OQ427348). Polyphasic identification indicated 100% similarity of FL1 and FL2 to F. luffae. A combined dataset of TEF1 and RPB2 was aligned using MAFFT v.7, and phylogenetic analysis was performed in MEGA v.7.0 using the maximum likelihood method. The cherry tomato isolates (FL1 and FL2) clustered together with the F. luffae reference strain NRRL31167 (100% bootstrap) and were identified on a morphological and molecular basis as F. luffae belonging to the Fusarium incarnatum-equiseti species complex. F. luffae was the only pathogen recovered from the infected fruit. To test for pathogenicity, healthy cherry tomato fruit were inoculated in a greenhouse (28°C, 12/12 h light/dark cycle, 90% relative humidity), six by wounded inoculation and six by nonwounded inoculation) with 10 μL conidial suspensions of isolate FL1 at 1 × 106 conidia/mL. Six wounded-treated cherry tomato fruit were used for the control. All cherry tomatoes were kept in a growth chamber at 28℃ with 90% relative humidity. After seven days, the inside of the wound inoculated fruit began to rot, expanding toward the surface and producing white mycelia. Two diseased cherry tomatoes were randomly selected for tissue isolation and F. luffae was re-isolated showing the same morphology as the FL1 isolate, thus fulfilling Koch's postulates. The nonwounded inoculated fruits and control cherry tomatoes remained asymptomatic with no pathogens recovered. This indicates that the wound is an important way for F. luffae to invade tomato, and fruit rot is caused by F. luffae's infection of tomato. To the best of our knowledge, F. luffae has caused fruit rot in muskmelon (Zhang et al. 2022), but this is the first report of fruit rot disease in cherry tomatoes caused by F. luffae in China. Since cherry tomatoes are an important commercial crop in China, F. luffae infection has the potential to pose a threat to the industry.

Global Scenario of Vegetable Fungal Diseases

Plant diseases threaten crop production and are responsible for considerable losses in vegetables worldwide. Every year diseases cause losses up to 40 to 60% in vegetable crops. Vegetable crops are comparatively more susceptible to various types of diseases inflicted by fungi, bacteria, virus, viroid, phytoplasma and nematodes. Genera of fungal pathogens viz. Alternaria, Aschochyta, Colletotrichum, Didymella, Phoma, Phytophthora, Pythium, Rhizoctonia, Sclerotinia and Sclerotium on vegetable crops increased tremendously. Emerging seed-borne and seed-transmitted fungal plant diseases became a major threat for the vegetable seed production system. Emerging diseases in vegetable crops like tomato (early and late blight, Fusarium wilt), chili and peppers (anthracnose, Phytophthora blight), brinjal (phomopsis blight, Alternaria leaf spot), cucurbits (Downey mildew, gummy stem blight, Fusarium wilt, fruit rot, anthracnose and Cercospora leaf spot), okra (Cercospora leaf spot), pea (powdery mildew, fusarium wilt, rust and root rot), French bean (Sclerotinia stem rot), cowpea/dolichos bean (anthracnose, Sclerotinia stem rot), cole crop (black rot, Alternaria blight, downy mildew) etc. The vegetable produce is spoiled by post-harvest pathogens and makes them unfit for human consumption and market due to the production of mycotoxins. Profiling, detection and diagnosis of vegetable pathogens (diseases) are essential for better understanding of pathogens and formulation of safe disease management strategy of vegetable crops. Therefore, there is an urgent need to wide array of diagnoses of pathogens of vegetables and their in-vitro testing towards chemo-sensitivity for formulation of safe management of vegetable diseases

Management of Fusarium fruit rot of Luffa cylindrical caused by Fusarium incarnatum and Fusarium chlamydosporum

Fusarium rot of Luffa cylindrical was detected at Kafr-El-Dawar province, Beheira governorate, North Egypt during the growing season 2021. Surveyed disease incidence at Beheira, Kafr El-Shekh and Sharkia governorate showed fruit rot incidences of 18.33%, 16.12%, and 12.42%, respectively. Two fungal isolates of Fusarium spp. were tested for their pathogenic ability to induce Fusarium rot disease and typical disease symptoms. The online Basic Local Alignment Search Tool for Nucleotide Sequences (BLASTN) was used for identification and confirmation of identity performance of Internal Transcribed Spacer (ITS) and DNA sequencing, then compared with sequences available in GenBank. The two fungal isolates were identified as Fusarium incarnatum and Fusarium chlamydosporum and registered under accession number MN480497 and MN480498, respectively. Under laboratory conditions, organic acids, essential oils and organic salts significantly inhibited the growth of the two fungal isolates. Complete inhibition of fungal growth (100%) was recorded at 0.75 g/l of Topsin-M70 (Thiophanate-methyl) and 0.5% of salicylic acid. Meanwhile, a moderate growth reduction was observed using benzoic acid, coumarine, and cinnamon, thyme oil and garlic oil treatments. Under field conditions for two successive seasons, similar trends were observed. Foliar sprays with the fungicide Topsin-M70 showed the largest reduction in disease expression. The highest fruit rot disease reduction was recorded for salicylic acid and coumarin treatments followed by thyme oil, benzoic acid and potassium carbonate, garlic oil, boric acid and sodium bicarbonate. We concluded that the treatments are effective, safe and cost-effective methods for the control of this pre-harvest fruit disease under field conditions.

Characterization and toxicological potential of Alternaria alternata associated with post-harvest fruit rot of Prunus avium in China.

Post-harvest fruit rot caused by Alternaria species is one of the most important threats to the fruit industry. Post-harvest rot on sweet cherry (Prunus avium) fruit was observed in the fruit markets of the Haidian district of Beijing, China. The fungal isolates obtained from the infected sweet cherry fruits matched the descriptions of Alternaria alternata based on the morphology and multi-gene (ITS, endo-PG, and Alta1) sequence analysis. Pathogenicity tests indicated that ACT-3 was the most virulent isolate, exhibiting typical post-harvest fruit rot symptoms. Physiological studies revealed that the optimal conditions for the growth of ACT-3 were temperature of 28°C, water activity of 0.999, and pH of 8 with 87, 85, and 86 mm radial growth of ACT-3 on a potato dextrose agar (PDA) medium, respectively, at 12 days post-inoculation (dpi). Moreover, the fungus showed the highest growth on a Martin agar medium (MAM) modified (85 mm) and a PDA medium (84 mm) at 12 dpi. The proliferation of the fungus was visualized inside the fruit tissues by confocal and scanning electron microscope (SEM), revealing the invasion and destruction of fruit tissues. Alternaria mycotoxins, tenuazonic acid (TeA), and alternariol (AOH) were detected in five representative isolates by HPLC analysis. The highest concentrations of TeA (313 μg/mL) and AOH (8.9 μg/mL) were observed in ACT-6 and ACT-3 isolates, respectively. This study is the first to present a detailed report on the characteristics and proliferation of A. alternata associated with sweet cherry fruit rot and the detection of toxic metabolites.

Rhizopus oryzae causes the leaf rot disease of Epimedium sagittatum in Guizhou, China.

Epimedium sagittatum is a collective term for herbaceous plants belonging to the family Berberidaceae. Their dried leaves and stems have significant therapeutic effects on tumor inhibition, hypertension control, and coronary heart disease (Ke et al. 2023; Zhao et al. 2019). In 2021 and 2022, plants with similar leaf rot symptoms ranging from 30% to 55% was observed on E. sagittatum in Congjiang County, Guizhou province. The initial symptoms of the disease manifest locally on the leaf, with yellowing on the surface edge of the affected tissue, browning in the middle part, and brown-white discoloration in the innermost part (Supplementary Figure S1B). As the disease progresses, the entire infected leaf gradually softens, while the veins remain intact (Supplementary Figure S1C). Ultimately, the leaf withers and dehisces. The nine samples with typical symptoms were collected from Congjiang County, Guizhou province (26.598°N, 106.707°E). Twenty-seven fungi were isolated, including ten isolates of Rhizopus and seventeen isolates of seven other genera. On isolate YYH-CJ-17 many sporangia were formed and turned to a brown-gray to black color on potato dextrose agar medium (PDA) after culturing 5 days under dark at 25 ℃ (Supplementary Figure S2A and S2B). The branches of mycelium were finger-shaped or root-shaped. The sporangium was spherical or nearly spherical, 60-250 µm in diameter, and sporangiospores were elliptical or spherical and 4-8 µm in diameter. The obtained 547 bp ITS fragment (accession OR225970) and 1231 bp EF-1α region (accession OR242258) from isolate YYH-CJ-17 were compared with NR database using the BLAST tool provided by NCBI, which revealed more than 99.5% identity (query cover more than 98%) with the sequences of ITS (accessions MF522822.1) and EF-1α (accession AB281541.1) of Rhizopus oryzae Went & H.C. Prinsen Geerlings (Gao et al. 2022; Zhang et al. 2022). The phylogenetic tree constructed with the ITS and EF-1α gene sequences demonstrates that strain YYH-CJ-17 clusters with R. oryzae in the same branch and the bootstrap value was greater than 99% (Supplementary Figure S3). Based on the morphological characteristics and ITS and EF-1a sequences, the isolate YYH-CJ-17 is identified as R. oryzae. Pathogenicity tests were performed on detached healthy leaves and living plants of E. sagittatum. Healthy leaves of E. sagittatum were subjected to inoculation with isolate YYH-CJ-17 with 5 × 105 CFU mL-1 concentration in sterile culture dishes. The progression of the disease was marked by the gradual softening of the infected leaves and the expansion of the lesions, which ultimately produced black-brown sporangium (Supplementary Figure S4A). Furthermore, the E. sagittatum living plants were sprayed with 5 × 105 CFU mL-1 conidial suspension of isolate YYH-CJ-17, with ddH2O as a negative control, and then were cultivated at 25℃ and 90% humidity for 21 days in the greenhouse. This assay found that the E. sagittatum leaves treated with isolate YYH-CJ-17 exhibited the same symptoms observed on plants in fields (Supplementary Figure S4B). The fungus re-isolated from the inoculated leaves were identified as R. oryzae by ITS sequencing and were blasted with NR database, which highest matched with the sequence of ITS (accessions MF522822.1) mentioned above, thus fulfilling Koch's postulates. R. oryzae has been identified as a causative agent of a diverse array of host diseases, including leaf mildew of tobacco, fruit rot of yellow oleander and pears, and soft rot of bananas (Farooq et al. 2017; Khokhar et al. 2019; Kwon et al. 2012; Pan et al. 2021). To the best of our knowledge, this is the first report of leaf rot on E. sagittatum caused by R. oryzae in China, which will provide clear prevention and management target for the leaf rot disease of E. sagittatum.

Effect of Different Culture Media on Growth and Establishment of Phomopsis vexans Inciting Fruit Rot of Brinjal (Solanum melongena L.)

The fruit rot caused by Phomopsis vexans (Sacc. & Syd.) Harter is a destructive disease of brinjal and is considered to be a major constraint for its production. The present study was carried out at the Centre of Excellence for Sanitary and Phytosanitary, Department of Plant Pathology, Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut, for the evaluation of different culture media on growth and development of Phomopsis vexans. The study showed the maximum radial growth on OMA (90.00 mm), followed by PDA (88.20 mm) and Sabouraud Dextrose Agar (80.16 mm). The pycnidial densities were recorded as excellent on OMA and TFA, good on PDA, SDA, BFA, and MEA, and poor on YMA and CMA media with brown to black colour, while on RSA and WA they were absent. The colony colour were observed on several cultural media with circular and irregular shapes. The maximum pycnidial size was recorded on TFA (454.35×316.18 µm), followed by SDA (415.60× 296.26 µm) and BFA (324.72×368.84 µm), whereas the minimum was recorded on CMA (289.48×128.52 µm), followed by MEA (308.66×140.25 µm). The α-conidia were noticed to be sub-cylindrical and elliptical in shape and largest conidia observed on TMA (10.6×2.6µm), followed by BFA (9.8×2.6µm), and the smallest on RSA (6.1×1.6µm). The number of conidia was recorded highest on OMA (29.) medium and lowest on RSA (12.63). The β-conidia were not observed in this study. The number of guttullae were recorded 1-2 in all tested culture media except TFA and SDA, which had 2-3.

Residue dissipation kinetics, safety evaluation and decontamination of Deltamethrin in tomato and the soil

Tomato powdery mildew, fruit rot, and twig blight are all managed with Deltamethrin. Its residues could still be present in the crops, posing a health risk. The pesticide residue analysis, dissipation rate, and safety assessments were thus examined in green tomatoes. The analytical method for residue analysis was validated according to international standards. Tomato fruits and soil were used to study the dissipation of Deltamethrin 100 EC (11% w/w) at 12.5 g a.i ha−1 for the recommended dose (RD) and 25.0 g a.i ha−1 for the double of the recommended dose (DD). Ethyl acetate was used to extract residues from tomato fruit, and PSA and magnesium sulphate were used for cleanup.The fruits had recoveries ranging from 83% to 93% and the soil sample from 81.67% to 89.6%, with the limit of detection (LOQ) estimated at 0.01 mg kg−1. The matrix effect (ME) was calculated to be less than 20% for the tomato fruits and the soil.Half-lives for RD and DD were 1.95 and 1.84 days, respectively. All sampling days for both doses had dietary exposures of residues below the maximum permissible intake (MPI) of 0.16 mg person−1 day−1. The most effective method of decontaminating tomato residue containing Deltamethrin is blanching.

First Report of Postharvest Fruit Rot Caused by Botrytis cinerea on Blue Honeysuckle (Lonicera caerulea L.) Fruit in China.

Blue honeysuckle (Lonicera caerulea L.) fruit is growing in popularity as a natural, functional 'super fruit', but its storage is challenged by pathogen infection. In June 2022, approximately 30% of 100 kg of blue honeysuckle fruits (cv. Lanjingling) obtained in Harbin, China (128.70°E, 44.87°N) showed postharvest fruit rot symptoms after 15 d of storage at 4°C, leading to whole fruit rotting with gray fungal growth (Fig.1 A). Small (1-2 mm) segments of infected tissue were obtained from 20 randomly selected fruits which were surface sterilized with 75% ethanol for 30 s and 5% sodium hypochlorite (NaOCl) for 3 min, rinsed three times with sterile distilled water, dried in paper towel, and plated in 9 cm Petri dishes containing potato dextrose agar (PDA). Five purified cultures were obtained and their front colonies were dark brown (Fig.1 C) on the PDA plates after 5 d at 25°C (Alam et al. 2019; Riquelme-Toledo et al. 2020). The conidia (n = 50) were single-celled, hyaline, either ellipsoid or ovoid, and measured 7.5-15.0 μm (11.7 μm average) × 6.0-11.4 μm (8.3 μm average). The conidiophores (Fig.1 E) were branched at the apex bearing bunches of conidia resembling grape clusters (Ellis 1971). For molecular confirmation, genomic DNA was extracted from a representative isolate LDGS-3 using the Ezup Column Fungi Genomic DNA Purification kit (Sangon Biotech, Shanghai, China). The internal transcribed spacer region (ITS, GenBank ON952502), heat shock protein (HSP60, GenBank OP039103), the second-largest subunit of RNA polymerase II (RPB2, GenBank OP186114) and glyceraldehyde 3-phosphate dehydrogenase (G3PDH, GenBank OQ658508) genes were partially amplified with the respective primers ITS1/ITS4, HSP60f/HSP60r, RPB2f/RPB2r, and G3PDH-F/G3PDH-R (Staats et al. 2005; White et al. 1990). BLAST analysis revealed that the sequences of the four genes showed 100% homology with the MH782039, MH796663, MN448501 and MH796662 sequences for isolates of Botrytis cinerea. Based on morphology and molecular characteristics, the isolate LDGS-3 was identified as B. cinerea. For pathogenicity, twenty healthy blue honeysuckle fruits (cv. Lanjingling) were superficially sterilized with 75% ethanol and washed with distilled water. Ten inoculated blue honeysuckle fruits, which were injected with 10 μL conidial suspension of isolate LDGS-3 (106 spores/mL) displayed fruit rot symptoms (Fig.1 B) inside 9 cm Petri dishes after 10 d at 4°C, while no symptoms were detected on ten fruits inoculated with sterile distilled water (Alam et al. 2019). The same isolate that was reisolated from infected fruits with the same morphological and molecular traits was also identified as B. cinerea, confirming Koch's postulates. B. cinerea was previously reported in Henan Province, China in hawthorn (Zhang et al. 2018). To our knowledge, this is the first report of postharvest fruit rot caused by B. cinerea on blue honeysuckle fruit in China, which will aid future management of this emerging postharvest disease.

The Pectinase Production Potential of Microorganisms Isolated from Agricultural Soil and Rotten Fruits in Shiraz, Southwest of Iran

The Pectinase Production Potential of Microorganisms Isolated from Agricultural Soil and Rotten Fruits in Shiraz, Southwest of Iran

First Report of Lasiodiplodia pseudotheobromae Causing Postharvest Decay of Strawberries in Florida

Postharvest decay of strawberry (Fragaria × ananassa Duch.) is a major factor causing fruit losses. Strawberries were obtained from various harvests at cooling facilities located in Dover and Plant City, FL during the 2018-19 and 2019-20 seasons. After the fruits were incubated at 22ºC for up to 5 days (d) to promote disease development, Lasiodiplodia decay was observed at up to 3% from some harvests, exhibiting gray mycelia on small lesions that gradually covered the whole fruit. The fungus was isolated onto potato dextrose agar (PDA). Five isolates (SBD18-14, SBD18-277, SBD18-279, SBD19-02 and SBD19-57) were characterized. Fungal mycelia were initially grayish white and then gradually changed to gray to dark gray on PDA at 25oC, and later produced black pigments (Fig. S1). Pycnidia were observed from inoculated strawberries at 14 d. Isolates shared similar conidia morphology: aseptate, hyaline, ellipsoid to ovoid, measuring L × W: 24.0-34.0 (28.3) × 13.0-16.0 (14.3) μm (n =100). Mature conidia were brown, one septate, measuring L × W: 25.0-33.0 (28.8) × 13.0-16.0 (14.5) μm (n =100). The isolates were identified as Lasiodiplodia spp. morphologically (Alves et al. 2008). DNA was extracted from fungal mycelia using an OmniPrep DNA extraction kit, and PCR amplification of ITS and EF1-α genes was performed following the conditions described by White et al. (1990) with some modifications using primers ITS1F-F/ITS4-R (Gardes and Bruns, 1993; White et al., 1990) and EF1-668-F/EF1-1251-R (Alves et al., 2008), respectively. The BLASTn in GenBank showed that the sequences obtained had 99.61 to 100% homology with those of ITS (EF622077) and EF1-α (EF622057) from L. pseudotheobromae CBS116459 (an ex-type strain) (Alves et al., 2008). Sequences of the isolates have been deposited in GenBank with accessions OP326017 to OP326021 for ITS, and OP356202 to OP356206 for EF1-α. Phylogenetic analysis showed that these isolates clustered in the same clade (bootstrap value at 64) with L. pseudotheobromae (Fig. S2). Two fungal inoculum types (mycelia and conidia), two fruit inoculation methods (injury and non-injury) and five fungal isolates were used for pathogenicity tests. Fungal mycelia (2-day-old) on PDA plug (5 mm) or 10 µL of conidial suspension (106 spores/mL) was placed onto each injury (1 x 1 mm in size) or a non-injury area on the surfaces of five strawberry fruits (cv. Florida Brilliance). PDA plug alone or water drops placed on injury or non-injury areas on fruits served as respective controls. Inoculated and control fruits were incubated in a covered plastic container with 100% RH at 22ºC. The experiment was repeated twice. Decay initially appeared as soft and lightly discolored tissue at inoculation areas 2 d post-inoculation (dpi) that extended quickly thereafter. Brown to dark lesions on both injury- and non-injury fruits inoculated with conidia or mycelia were observed at 3 dpi. Decay and gray mycelia gradually developed over the whole fruit at 6 dpi, and pycnidia were observed after 14 dpi (Fig. S1). Disease incidence of 100% was observed on all tests. Control fruits did not develop decay. The results indicate that these isolates are pathogenic to strawberries and infect fruit via both non-injured and injured fruit surfaces. The inoculated fungal isolates were re-isolated, thus, fulfilling Koch’s postulates. L. theobromae, Neofusicoccum parvum/N. ribis species complex causing strawberry fruit rot in Florida fields was reported (Oliveira et al., 2019), but not L. pseudotheobromae. To our knowledge, this is the first report of postharvest decay caused by L. pseudotheobromae A.J.L. Phillips, A. Alves & Crous on strawberries in Florida and in the USA, and it should be considered in strawberry disease management.

First report of Diplodia bulgarica causing black canker on apple in California.

California is the sixth largest apple-producing state in the United States with a production that reached 4,654 ha in 2021. During the late winter of 2023, black canker symptoms were observed on branches of 'Gravenstein' apple (Malus domestica) in two commercial orchards in Sonoma County, California. The prevalence of symptomatic trees ranged from 10 to 30%. External symptoms included charcoal looking-cankers with the bark peeling off from the primary and secondary branches. Internally, cankers were dark brown in color with a hard consistency. Pycnidia were observed on the surface of older cankers. Fungal isolations were performed from disinfected (70% ethanol, 30 s) symptomatic branch samples (n = 15). Small wood pieces (5 mm length) were taken from the margin of diseased and healthy tissues, and placed on potato dextrose agar acidified with 92% lactic acid at 0.5 mL per liter (APDA). Plates were incubated at room temperature (20-22 °C) for 7 days. Colonies of Botryosphaeriaceae species (Phillips et al. 2013) (n = 12) were consistently recovered and pure cultures were obtained by transferring a single hyphal tip onto fresh APDA. Colonies were light gray with irregular margins. To induce pycnidia formation, two isolates (UCD11350 and UCD11351) were grown on pistachio leaf agar for 21 days. Conidia (n = 50) were thick-walled and ovoid in shape, initially hyaline, then turned pale brown and dark brown at maturity, and some of them became 1-septate, ranging from 18.9 to 24.0 (21.9) × 11.5 to 14.7 (13.4) µm. Isolates were identified by sequencing a partial region of the beta-tubulin (tub2) gene using the primers Bt2a/Bt2b (Glass and Donaldson 1995). BLAST searches on NCBI GenBank revealed 99.5 % identity with the Diplodia bulgarica ex-type (CBS 1245254). To confirm the identity, the rRNA internal transcribed spacer (ITS) and the translation elongation factor 1-alpha (tef1) were also sequenced using ITS5/ITS4 (White et al. 1990), and EF1-688F/EF1-1251R (Alves et al. 2008), respectively. A maximum parsimony multi-locus phylogenetic analysis clustered Californian isolates with reference strains of D. bulgarica. Sequences were deposited in GenBank (nos. OR631209 to OR631210, OR637361 to OR637362, OR637363 to OR637364 for ITS, tub2, and tef1, respectively). Pathogenicity tests were conducted on 2 to 3-year-old branches (n = 5) of over 20-year-old trees by inserting a 5-mm segment of a toothpick, completely colonized with each of the two isolates mentioned above, into a 1-mm-diameter hole made with a sterile drill bit. The same number of branches where mock inoculated with a non-colonized toothpick as negative control. The experiment was performed twice. After ten weeks, inoculations resulted in dark brown necrotic lesions that ranged from 54.0 to 59.8 mm in length. Negative controls remained asymptomatic. Koch's postulates were fulfilled by successfully recovering the isolates from the lesion margins, which were confirmed by morphology. Diplodia bulgarica was first described affecting M. sylvestris in Bulgaria (Phillips et al. 2012), and then detected on M. domestica causing cankers in Iran (Abdollahzadeh 2015), India (Nabi et al. 2020), Germany (Hinrichs-Berger al. 2021) and Türkiye (Eken 2021). The pathogen was also identified causing postharvest fruit rot (Eken 2022). To our knowledge, this is the first report of D. bulgarica causing branch canker on apple in California, which provides important information for developing detection and control strategies.

Quantitative Trait Loci Analysis and Marker Development for Fruit Rot Resistance in Cranberry Shows Potential Genetic Association with Epicuticular Wax.

Fruit rot is a fungal disease complex that threatens cranberry yields in North American growing operations. Management of fruit rot is especially difficult because of the diversity of the infecting fungal species, and although infections take place early in the season, the pathogens usually remain latent in the ovary until the fruit ripen. Control methods heavily rely on fungicide applications, a practice that may be limited in viability long term. Breeding for fruit rot resistance (FRR) is essential for sustainable production. It is likely that field resistance is multifaceted and involves a myriad of traits that fortify cranberry plants against the biotic and abiotic stresses contributing to fruit rot. In this study, we identified quantitative trait loci (QTL) for FRR in a segregating population. Interestingly, a QTL associated with resistance was found to overlap with one associated with fruit epicuticular wax (ECW). A single-nucleotide polymorphism genotyping assay successfully identified accessions that exhibit the desired phenotypes (i.e., less rot and more ECW), thus making it a useful tool for marker-assisted selection. Candidate genes that may contribute to FRR and ECW were also identified. This work will expedite breeding for improved cranberry fruit quality.

Klasifikasi Buah dan Sayuran Segar atau Busuk Menggunakan Convolutional Neural Network

Fresh fruits and vegetables contain many nutrients, such as minerals, vitamins, antioxidants, and beneficial fiber, superior to those found in rotten or almost rotten produce. On the other hand, fruits and vegetables that are nearly spoiled or already rotten have significantly lost their nutritional value. Rotten produce also harbors bacteria and fungi that can lead to infections and food poisoning when consumed. Convolutional Neural Network (CNN) offers a programmable solution for classifying fresh and rotten fruits and vegetables. Image processing using the TensorFlow library is employed in this classification process. During testing on the training data, the CNN achieved an accuracy of 90.42%. In comparison, the validation accuracy reached 94.21% when using the SGD optimizer, 20 epochs, a batch size 16, and a learning rate of 0.01. For the testing data, the accuracy obtained was 80.83%.