First Report of Fruit Rot Caused by Fusarium luffae in Muskmelon in China

Muskmelon (Cucumis melo L.) is one of the most widely cultivated and economically important fruit crops in the world. However, many pathogens can cause decay of muskmelon fruit, including Fusarium spp.. Fusarium spp. are the most important pathogen, affecting muskmelon fruit yield and quality (Wang et al. 2011). In August 2020, fruit rot symptoms were observed on ripening muskmelons (cv. Tianbao) in several fields in Jiyang District, Jinan City of Shandong Province, China. The incidences of infected muskmelon ranged from 15% to 30% and caused an average 20% yield loss. Symptoms appeared as pale brown, water-soaked lesions that were irregular in shape, with the lesion sizes ranging from a small spot (1 to 2 cm) to decay of the entire fruit. The core and surface of infected fruit were colonized and covered with white mycelia. Two infected muskmelons were collected from two fields, 3.5 km apart. Tissues removed from inside the infected fruit were surface disinfected with 75% ethanol for 30 s, and cultured on potato dextrose agar (PDA) at 25°C in the dark for 5 days. Four purified cultures were obtained using the single spore method. On carnation leaf agar (CLA), 3 to 5 septate, falcate, with a pronounced dorsiventral curvature macroconidia with tapered apical cell, and foot-shaped basal cell, measuring 20 to 40 × 3.5 to 4.5 μm. Microconidia and chlamydospores were not observed. These morphological characteristics were consistent with the description of F. luffae (Wang et al., 2019). Because these isolates had similar morphology, two representative isolates (XP11 and XP12) were selected for multilocus phylogenetic analyses. DNA was extracted from the representative isolates using a CTAB method. Nucleotide sequences of the internal transcribed spacers (ITS) (White et al. 1990), calmodulin (CAM), RNA polymerase II second largest subunit (RPB2), translation elongation factor 1-α gene (TEF1) (Xia et al. 2019) were amplified using specific primers, sequenced, and deposited in GenBank (ITS: MW391509 and MW391510, CAM: MW392789 and MW392790, RPB2: MW392797 and MW392798, TEF1: MW392793 and MW392794). Alignments of a combined dataset of ITS, CAM, RPB2 and TEF1 were made using MAFFT v. 7, and phylogenetic analyses were conducted in MEGA v. 7.0 using the maximum likelihood method. The muskmelon isolates (XP11 and XP12) clustered together with the F. luffae reference strain LC12167 (99% bootstrap). To perform a pathogenicity test, 10 μl of conidial suspensions (1 × 106 conidia/ml) were injected into each muskmelon fruit using a syringe, and the control fruit was inoculated with 10 μl of sterile distilled water. There were ten replicated fruits for each treatment. The test was repeated three times. After 7 days at 25°C, the interior of the inoculated muskmelons begun to rot, and the rot lesion expanded from the core towards the surface of the fruit, then white mycelia were produced on the surface. Ten isolations were re-isolated from the infected tissues and confirmed to fulfill Koch’s postulates. No symptoms were observed on the control muskmelons. To our knowledge, this is the first report of fruit rot caused by F. luffae in muskmelon in China. Considering the economic value of the muskmelon crop, correct identification can help farmers select appropriate field management measures for control of this disease.

First Report of Fusarium meridionale Causing Stalk Rot of Ryegrass in China

Members of the Fusarium graminearum species complex (FGSC) are the main causing agents of head blight, seedling blight, or stalk rot in wheat and other cereals worldwide. Surveys on species composition and mycotoxin production of FGSC populations have mainly focused on food crops such as wheat, maize, and barley, but little is known about the identity of FGSC pathogens present in pasture grass. In April 2021, a survey of grass diseases in the Hongya County (29.90661 N; 103.37313 E) in Sichuan Province was conducted to understand the etiology of stalk rot in perennial ryegrass (Lolium perenne). It was observed in several pastures that about 10% of yield loss in perennial ryegrass was caused by stalk rot. Affected plant stalks were brown to dark brown in colour and appeared soggy. As infections continued or under conditions of high humidity, some plant stalks also became flattened. Perennial ryegrass samples with symptoms of stalk rot or browning of the stem were collected. Symptomatic tissues were cut into short segments (approximately 5 mm), surface-sterilized in 3% sodium hypochlorite solution for 2 min, rinsed three times with sterile distilled water, air dried, plated onto potato dextrose agar (PDA), and then incubated in the dark at 28 °C. After 3 to 5 days, Fusarium-like fungal colonies with reddish-orange mycelium were collected and transferred to new PDA plates for further purification, and the purified cultures were obtained by single spore isolation. Four uniform isolates were obtained and their colonies on PDA resembled typical FGSC colonies (Leslie and Summerell 2006; O’Donnell et al. 2004). Colonies had an average radial growth rate of 8.5 to 11.0 mm/day at 28 °C in the dark on PDA. Conidial characteristics were studied on Spezieller Nährstoffarmer agar (SNA) as described by Wang et al. (2014). Macroconidia were falcate to almost straight, usually with parallel dorsal and ventral lines, 3- to 5-septate, 20.65 to 55.22 μm in length (average 39.16 μm), and 2.38 to 6.93 μm in width (average 4.42 μm) (n = 200). No microconidia were observed. The pathogenicity of the isolated Fusarium strains was then tested on healthy perennial ryegrass (variety Changjiang 1). Ryegrass plants grown for 2 months were inoculated by punching a hole in the stem using a sterile toothpick, followed by an injection of 20 μL macroconidia suspension at a concentration of 105 spores/mL. Ryegrass stems treated with water served as the control. Twenty plants were included in each treatment. After inoculation, the plants were grown in a growth chamber at 25 °C and 90% humidity for 24 h. Stalk tissues at the wound site turned brown after 3 days and the brown area then extended to regions above and below. No symptoms were observed in the water-treated controls. As well, the same pathogen was reisolated from the infected grass stems, but not from the controls. Thus, the isolated Fusarium spp. are a cause of stalk rot in perennial ryegrass based on the fulfillment of Koch’s postulates. To identify the Fusarium spp. to species level, portions of the translation elongation factor 1-α (TEF) gene sequences from all four strains were amplified and sequenced as described by Wang et al. (2015). The obtained sequences were identical, and a sequence of isolate SC1 was submitted to GenBank (accession no. MZ964308). BLASTn searches were conducted on the TEF sequence (607 bp) in two databases, revealing it had 100% similarity to the sequence of Fusarium meridionale strain DS27 (accession no. MN629330) in NCBI and strain NRRL28723 from FUSARIUM-ID (http://isolate.fusariumdb.org/). A concatenated four-gene phylogeny (supplementary figure) resolved SC1 and the type specimens of F. meridionale (NRRL28723, 29010, and 28436) in a monophyletic clade with 100% bootstrap support, confirming that the strain SC1 belongs to F. meridionale. Finally, trichothecene productions of F. meridionale strains were evaluated using rice cultures kept at 28 °C in the dark for two weeks, as described by Desjardins and Proctor (2011). LC-MS/MS analysis indicated that the fungus could produce NIV and 4ANIV in rice cultures with average concentrations of 1400.44 and 3144.10 μg/kg, respectively. To the best of our knowledge, this is the first report of F. meridionale causing disease in perennial ryegrass in China. Further research will be necessary to determine its distribution, aggressiveness, and trichothecene production.

Antagonistic screening of Trichoderma spp. isolated from patchouli rhizosphere

The objective of this research was to determine the antagonistic activity of Trichoderma spp. isolated from patchouli rhizosphere (Pogostemon cablin Benth.). Another objective was to perform antagonistic screening of these fungi to inhibit the growth of the wilted pathogen Fusarium spp. In vitro research was conducted in the Laboratory of Plant Pathology, Universitas Syiah Kuala, from January to June 2020. The study used a completely randomised design with five treatments and three replications. The antagonistic screening was carried out by using the dual culture method of Trichoderma spp. against Fusarium spp. with the medium of Potato Dextrose Agar (PDA). The result showed that five isolates of Trichoderma have different antagonistic percentages in inhibiting the Fusarium. The highest antagonistic activity was found from isolate 2 and the lowest value was shown by isolate 3.

Enzyme Linked Immunosorbent Assay for Fumonisin B1 Detection in Local Corn Seeds from Baghdad-Iraq

Fungi produce a series of toxic compounds on corn, especially Fumonisin B1 (FB1) toxin produced by Fusarium spp. and promoting cancer activity in humans and animals. This study aimed to the isolation and identification of fungi associated with local corn seeds and the detection for the presence of FB1 by using ELISA technique. Thirty samples of corn ears were collected from silos and markets in Baghdad city during the period from November 2018 to March 2019. The present study found that Fusarium was the dominant isolate among fungi in terms of the relative density 57.07%, followed by Aspergillus 31.17%, Rhizopus 3.36%, Alternaria 2.88%, Mucor 2.16%, Penicillium 1.92%, Trichothecium 0.96%, and Helminthosporium 0.48%. FB1 was detected in all samples of the silos and markets with a concentration range of 13.69 - 175.54 µg/kg. There were no significant differences in FB1concentration among samples collected from the silos and markets. Also, no relationship was found between the number of infected seeds by Fusarium spp. and FB1concentrations.

Enzymatic Bioprospecting of Fungi Isolated from a Tropical Rainforest in Mexico

The humid tropical environment provides an ideal place for developing a high diversity of plants; this is why it is an interesting site for the enzymatic bioprospecting of fungi that are responsible for the recycling of organic matter in an efficient and accelerated way and whose enzymes could have multiple biotechnological applications. For this study, 1250 isolates of macroscopic and microscopic fungal morphotypes were collected from soil, leaf litter, and wood. One hundred and fifty strains (50 from each source) were selected for the enzymatic screening. From the first phase, 51 strains with positive activity for laccase, protease, amylase, xylanase, and lipase enzymes were evaluated, of which 20 were isolated from leaf litter, 18 from the soil, and 13 from wood. The 10 best strains were selected for the enzymatic quantification, considering the potency index and the production of at least two enzymes. High laccase activity was detected for Trametes villosa FE35 and Marasmius sp. CE25 (1179 and 710.66 U/mg, respectively), while Daedalea flavida PE47 showed laccase (521.85 U/mg) and protease activities (80.66 U/mg). Fusarium spp. PH79 and FS400 strains had amylase (14.0 U/mg, 49.23 U/mg) and xylanase activities (40.05 U/mg, 36.03 U/mg) respectively. These results confirm the enzymatic potential of fungi that inhabit little-explored tropical rainforests with applications in industry.

The occurrence of Fusarium species in white spears of Asparagus officinalis

The aim of the study was to determine the species composition of fungi of the Fusarium genus found on white Asparagus officinalis spears, depending on the presence or absence of disease symptoms, age of the plantation, date of harvest and the place from which samples were collected for mycological analysis. Additionally, the pathogenicity of selected Fusarium spp. isolates was determined. A. officinalis L. was cultivated for white spears. The research was conducted on the German dioecious cultivar ‘Eposs’. Samples of A. officinalis spears for tests were collected from two plantations. Six species of fungi of the Fusarium genus were identified in the asparagus spears: F. culmorum, F. equiseti, F. oxysporum, F. proliferatum, F. solani, and F. fujikuroi. Among the Fusarium species colonizing Asparagus officinalis the greatest threat is F. oxysporum. Always there are more isolates in the spears with the symptoms, in epidermis. The late harvest date favors the development of fusariosis. This means that the spears harvested at the latest date (late June) are the most heavily colonised by fungi. The isolates of fungi of the Fusarium genus collected from the spears exhibit pathogenicity against A. officinalis plants.

Malignant Keratitis Caused by a Highly-Resistant Strain of Fusarium Tonkinense from the Fusarium Solani Complex

Fusarium spp. are moulds ubiquitously distributed in nature and only occasionally pathogenic for humans. Species of the Fusarium solani complex are the predominant keratitis-inducing pathogens, because they are endowed with proper virulence factors. These fungi can adhere to the cornea creating a biofilm and, with the help of enzymes and cytotoxins, penetrate the cornea. Whereas an intact cornea is hardly able to be invaded by Fusarium spp. in spite of appropriate virulence factors, these opportunistic fungi may profit from predisposing conditions, for example mechanical injuries. This can lead to a progressive course of corneal infection and may finally affect the whole eye up to the need for enucleation. Here, we present and discuss the clinical, microbiological and histopathological aspects of a particular case due to Fusarium tonkinense of the Fusarium solani complex with severe consequences in a patient without any obvious predisposing factors. A broad portfolio of antifungal agents was applied, both topically and systemically as well as two penetrating keratoplasties were performed. The exact determination of the etiologic agent of the fungal infection proved likewise to be very challenging.

BIODIVERSIDADE DE BACTÉRIAS ANTAGÔNICAS A FUNGOS ASSOCIADOS À SINDROME DA MORTE DO CAPIM-MARANDU (Brachiaria brizantha)

A síndrome da morte do capim-marandu (Brachiaria brizantha) é um dos principais problemas na degradação das pastagens no bioma Amazônia. A morte das forrageiras ocorre durante a época chuvosa por suscetibilidade a fitopatógenos. Assim, objetivou-se estudar sistemas de produção e selecionar bactérias antagônicas a Fusarium spp., um dos agentes fitopatogênicos. Os isolados bacterianos foram obtidos de 10 sistemas de produção, mata nativa e pousio, na chuva e na seca. Realizou-se a caracterização molecular de isolados por sequenciamento. Dois isolados de Fusarium foram testados. Para o patógeno 1, na época da chuva, 7,5% dos isolados apresentaram antagonismo enquanto na seca foi 15%. Para o patógeno 2, nem todos os isolados positivos para o patógeno 1 foram eficientes no controle, demonstrando a ocorrência de interações entre isolados bacterianos e Fusarium spp. Na chuva, para o patógeno 1, a mata apresentou maior número de isolados positivos (20%). Na seca, os tratamentos mata, 1-Floresta e 8-integração lavoura floresta para o patógeno 1, foram os que apresentaram maior número de positivos (25%). Na identificação molecular, 8 foram os gêneros bacterianos encontrados: Burkholderia, Bacillus, Brevibacillus, Streptomyces, Pseudomonas, Escherichia, Paenibacillus e Pandoraea, em que a maioria é descrita como capaz de controlar Fusarium spp. in vitro. Palavras-chave: Burkholderia; controle biológico; integração lavoura-pecuária-floresta. Biodiversity of bacteria antagonic to fungi associated with syndrome's death palisadegrass (Brachiaria brizantha) ABSTRACT: The death syndrome of marandu grass (Brachiaria brizantha) is one of the main problems in the degradation of pastures in the Amazon biome. Plant death occurs during the rainy season due to susceptibility to phytopathogens. Thus, the objective was to study production systems and select bacteria antagonistic to Fusarium spp., one of the phytopathogenic agents. Bacterial isolates were obtained from 10 production systems, native forest and fallow, in rain and drought season. Molecular characterization of isolates was carried out by sequencing. Two Fusarium isolates were assessed. For pathogen 1, in the rainy season, 7.5% of the isolates showed antagonism, while in the dry season it was 15%. For pathogen 2, not all isolates positive for pathogen 1 were efficient in the control, showing correlations between bacterial isolates and Fusarium spp. In the rain, for pathogen 1, the forest showed the highest number of positive isolates (20%). In the dry season, the treatments forest, 1-Forest and 8-plant-forest integration for pathogen 1, were ones with the highest number of positives (25%). In the molecular identification, 8 bacterial genera were found: Burkholderia, Bacillus, Brevibacillus, Streptomyces, Pseudomonas, Escherichia, Paenibacillus and Pandoraea, most of which are described as able to controlling Fusarium spp. in vitro. Keywords: Burkholderia; biological control; integrated crop-livestock-forest.