First report of Colletotrichum plurivorum and Colletotrichum truncatum causing anthracnose on melon plants in Brazil.

Melon (Cucumis melo L.) is an economically important crop in Brazil, with an annual production of 699.281 tons (FAO 2024). Fungal diseases are one of the biggest problems in melon production, and melon growers in northeastern Brazil have reported over 80% of plants showing anthracnose symptoms in the fields during rainy seasons. Plants were wilted, displaying brown necrotic lesions and water-soaked spots with yellowish edges on the leaves and vines. Melon fruits displayed necrotic lesions on the outside. From June 2022 to June 2023, melon leaves (varieties Yellow, Galia, and Cantaloupe) from anthracnose-symptomatic plants were collected in four melon farms located in the municipalities of Afonso Bezerra, Mossoró, Tibau, and Upanema in the state of Rio Grande do Norte. Small fragments of symptomatic leaves were disinfected in 70% ethanol (30 sec) and 2.5 % sodium hypochlorite (1 min), rinsed in sterile distilled water, and plated on PDA Petri dishes with tetracycline (0.05g/liter). Plates were maintained in a bio-oxygen demand incubator (BOD) for 3 days at 28 ± 2 °C, under a 12 hr photoperiod. Eleven representative fungal colonies resembling Colletotrichum spp. were selected and monosporically grown on PDA for seven days for morphology, pathogenicity, and molecular analyses.ight colonies showed pinkish-dark brown with acervuli in the center and cottony mycelium, and showing black edges in some isolates, resembling C. plurivorum (Zhang et al. 2023). Conidia from those colonies were hyaline, cylindrical with obtuse ends, and 17.76 x 7.06 µm, n= 50. Three colonies developed pinkish-gray mycelia with numerous black microsclerotia, and the conidia were hyaline, falcate, and 27.38 x 4.10 µm, n= 50, resembling C. truncatum (Yu et al. 2023). The total DNA of the eleven isolates was extracted, and the internal transcribed space (ITS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), actin (ACT), ß-tubulin (TUB), and chitin synthase 1 (CHS-1) regions were partially amplified by PCR. Amplicons were sequenced and deposited to Genbank (Table eXtra1). A phylogenetic tree was built with the Maximum likelihood method with the concatenated sequences of the five partial gene sequences on Software MEGA (Version 11.0.10) (Tamura et al. 2021). The isolates CML5, CML8, CML9, CML10, CML11, CML14, CML15, and CML25 were grouped with Colletotrichum plurivorum CBS 125474 (orchidearum complex), and the isolates CML26, CML27 and CML28 with Colletotrichum truncatum CBS 15:35 (truncatum complex) with 87 % e 97 % of Bootstrap support, respectively. C. plurivorum was detected in four farms visited (we selected two representative isolates per farm), while C. truncatum isolates were all from the farm in Afonso Bezerra municipality. A pathogenicity test was performed following the method of Baishuan et al. (2023), micro-injuries were made in leaves of melon seedlings 'Goldex Yellow' and inoculated with a spore suspension of colonies with seven days of growth (106 spore/mL) of each isolate and sprayed to the point of dripping. Sterile water was used as mock. After nine days, anthracnose symptoms similar to those observed in the field were seen in all inoculated leaves, while no symptom was observed in the leaves of the mock plants. The pathogens were reisolated and their identification was confirmed by morphology and sequencing. Five seedlings were inoculated per isolate and mock, the assay was repeated, and the same results were observed. The species C. plurivorum has already been reported to cause disease in Cucumbers in Brazil (Silva et al. 2023) and C. plurivorum and C. truncatum in Citrullus lanatus in China (Guo et al. 2022). To the best of our knowledge, this is the first report of C. plurivorum and C. truncatum causing anthracnose in melon plants in Brazil.

Herbicide Leaching in Soil with Different Properties: Perspectives from Commercial Formulations and Analytical Standards.

The leaching of herbicides into the soil is essential to control germinating seeds and parts of vegetative weeds. However, herbicide transportation to deeper soil layers can result in groundwater contamination and, consequently, environmental issues. In this research, our objective was to investigate differences in herbicide leaching between commercial formulations and analytical standards using three different soils. Leaching experiments were carried out for diuron, hexazinone, and sulfometuron-methyl herbicides isolated and in binary and ternary mixtures. The herbicide residue quantification was performed by ultra-high-performance liquid chromatography coupled to a mass spectrometer (LC-MS/MS). Diuron had less mobility in soils and was retained in the most superficial layers. Hexazinone and sulfometuron-methyl were more mobile and leached into deeper layers. The leaching process was more intense for hexazinone and sulfometuron-methyl. The additives present in the commercial formulation favored the leaching in soils of diuron, hexazinone, and sulfometuron-methyl herbicides isolated and mixture compared to the analytical standard. This fact highlights the importance of considering these effects for the positioning of herbicides in the field to increase the efficiency of weed control and minimize the potential for environmental contamination.

First report of Lasiodiplodia brasiliensis causing root rot in melon plants in northeastern Brazil.

Melon (Cucumis melo L.) is the second most exported fruit in Brazil with an annual production of 27.5 million tons (FAO 2023). From September 2019 through February 2020, 50-day-old melon plants started showing root rot symptoms (dark-brow necrotic zones in their roots that extended to the collar zone) in northeastern Brazil, 30% of the plants in the fields were affected by the disease. The fields are in clay soil where melon, in monoculture, is produced all year long with three cycles of the culture per year. A total of 132 samples from "Yellow" and "Cantaloupe" cultivars were collected from four melon fields (4°59'45.3"S, 37°33'39.7"W; 4°57'10.2"S, 37°31'37.1"W; 5°38'17.9"S, 37°56'27.7"W; and 5°00'25.5"S, 37°23'55.3"W). Small pieces of diseased tissues were surface disinfested in 70% ethanol for 30 sec, in 2% sodium hypochlorite for 1 min, washed in sterilized distilled water, plated on a PDA Petri dishes with tetracycline (0.05g/L), and incubated for seven days at 28 ± 2 ºC. Nine representative isolates were selected for downstream analysis. Colonies were white and later became dark gray, pycnidia and conidia were produced after 30 days ofncubation at 25°C under near-UV light in water-agar medium. Conidia were hyaline when immature and dark brown when mature, ranging from cylindrical subovoid to ellipsoidal and septate to non-septate, and with an average size of 12.54 to 21.97 µm. The colonies were morphologically identified as Lasiodiplodia sp. (Phillips et al. 2013). Total DNA from the isolates was extracted and the ITS, TUB, and TEF-1α genes (Jayawardena et al. 2019) were partially amplified by PCR, Sanger sequenced, and deposited in Genbank: ITS (OM102511 to OM102520), TUB (OR062087 to OR062094 and OR062095), and TEF-1α (OP536826 to OP536835). Blastn analysis of the partial sequences ITS (519bp), TUB (388bp), and TEF-1α (315bp) showed 100% nucleotide similarity of the isolates with sequences of L. brasiliensis and L. theobromae from the GenBank. A phylogenetic tree was constructed using the Maximum Parsimony Analysis method. All nine isolates were grouped into the L. brasiliensis clade with 71% bootstrap support, confirming the isolates's identity. Pathogenicity assays were conducted in a greenhouse using the wooden toothpick inoculation method (Nogueira et al. 2019). "Goldex" Yellow melon seedlings were used in a completely randomized experimental design, with 10 treatments (9 isolates + Mock) and six replicates, with one plant per pot. Plants were inoculated 15 days after sowing, and disease severity was evaluated 50 days after inoculation. All nine isolates caused symptoms in the assessed melon plants. The fungus was reisolated from the lesions and looked morphologically identical to the inoculated fungus, fulfilling Koch's postulates. The pathogenicity test was repeated and yielded similar results. All samples in this study were provided by melon growers who were concerned about the high incidence of root rot disease in their plantations. More research needs to be conducted to determine the epidemiology and the extension of the economic impact caused by this pathogen to melons to develop strategies for disease control to properly assist the growers's concerns. This pathogen has been reported to cause disease in other crops in Brazil, e.g., watermelon (Alves et al. 2023) and apples (Martins et al. 2018). However, to the best of our knowledge, this is the first report of L. brasiliensis causing root rot in melons in Brazil.

First Report of Cladosporium tenuissimum causing spot diseases on leaves and fruits of cucurbits in Brazil.

Cucurbitaceae crops are widely cultivated in the Northeast region of Brazil, which is the biggest producer of melon and watermelon in the country (Oliveira, 2020). Between November and December 2020 leaves of pumpkins (Cucurbita maxima L.) and watermelon (Citrullus lanatus L.), and leaves and fruits of melon plants (Cucumis melo L.) were collected with moderate to severe necrotic, irregular, and brown lesions from farms in the state of Rio Grande do Norte, Brazil. Fragments of diseased tissues were cut into small pieces and surface disinfested in 70% ethanol for 30 seconds, then in 2% sodium hypochlorite for 1 minute, and washed in sterile distilled water. Disinfested pieces of tissue were plated on potato dextrose agar (PDA) and incubated for seven days in the dark at 28 ± 2 °C. A total of 12 fungal isolates (four from pumpkins, one from watermelon, and seven from melons) were isolated from leaves and symptomatic fruits. All isolates in this study shared similar morphological characteristics. The colonies were dark gray to olive green in color with a velvety texture and surrounded by gray-white hyphae. The conidiophores were erect, tall, dark, and irregularly branched at the apex containing dark conidia, with 0 to 3 septa, variable in shape and size, forming chains that were often branched, globose, or subglobose with 3 to 4.5 µm in diameter. DNA from each isolate was extracted using the SDS method (Smith et al., 2001) and submitted to PCR amplification of the ITS and TEF1α regions with the primers ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively. The amplicons were sequenced and deposited in GenBank: ITS (OP493545-OP493556) and TEF1α (OP536836-OP536847). Blastn analysis of the ITS and TEF1α partial sequences revealed that all 12 isolates belong to the species Cladosporium tenuissimum, with 100% nucleotide similarity with sequences of many C. tenuissimum isolates deposited in GenBank. A phylogenetic tree was constructed using the Maximum Parsimony Analysis, with the concatenated sequences (ITS-TEF1α) on MEGAX software (version 11.0.8) (Tamura et al, 2018). All 12 isolates clustered in the same clade and were closely related to isolates A2PP5, A3I1, and XCHK2 with the respective accession numbers KU605789.1, KU605790.1, and MG873071.1 from GenBank, with 99% bootstrap support. The pathogenicity of the 12 isolates was evaluated in pumpkin and melon plants in a greenhouse. Spore suspensions (10 6 conidia/ml -1) were sprayed on the leaves of healthy seedlings until runoff, only water was sprayed on control plants as the mock, and five seedlings of each crop (melon and pumpkin) were inoculated in each treatment. All plants were covered with plastic bags for two days. Spots, similar to those observed on diseased plants in the field, developed on the inoculated leaves (after seven days from the inoculation day, no symptoms were observed on plants from the mock treatment) and the fungal morphology was identical to that observed on the originally diseased leaves, fulfilling Koch's postulate. The pathogenicity test was repeated and yielded the same results. The fact that all 12 isolates were pathogenic on pumpkin and melon leaves, indicates that many Cucurbits are susceptible to C. tenuissimum infection. Many growers in the region are reporting similar symptoms in their melon plantations and it appears that the disease incidence is getting more severe year after year, based on growers's reports. Therefore, more research needs to be conducted to determine the epidemiology and the extension of the economic impact caused by this pathogen to Cucurbits to develop strategies for disease control. To the best of our knowledge, this is the first report of C. tenuissimum causing disease in Cucurbits in Brazil.

Detection of Mycobacterium leprae DNA in clinical and environmental samples using serological analysis and PCR.

BACKGROUND: Leprosy is a chronic infectious disease caused by Mycobacterium leprae and persists as a serious public health problem in Brazil. This microorganism is inculturable, making it difficult to diagnose and elucidate details of its transmission chain. Thus, this study aimed to analyze the dynamics of environmental transmission of M. leprae in a case-control study in the city of Mossoró, Brazil. METHODS AND RESULTS: Data of clinical, epidemiological, bacilloscopic, and serological evaluation of 22 newly diagnosed patients were compared, with molecular results of detection of specific genome regions RLEP and 16S rRNA of M. leprae in samples of the nasal swab, saliva, and house dust of these individuals and their controls (44 household contacts and 44 peridomiciliar contacts). The rapid serological tests evaluated, ML flow (IgM ND-O-BSA) and OrangeLife® (IgM and IgG anti NDO-LID 1) showed similar results, with greater positivity among paucibacillaries by OrangeLife® (54.5%). Positivity for nasal swab and saliva in multibacillary patients with RLEP primer was 16.7% and 33.3%, respectively. There was no detection of bacterial DNA in house dust or among paucibacillaries. The OrangeLife® test indicated that the lower the amount of windows, the more transmission in the house (3.79 more chances). Having a history of leprosy cases in the family increased the risk by 2.89 times, and being over 60 years of age gave 3.6 times more chances of acquiring the disease. PCR positivity was higher among all clinical samples using the M. leprae RLEP region than 16S rRNA. CONCLUSIONS: In this study, the serological and PCR analysis were capable of detecting M. leprae DNA in clinical samples but not in the environmental samples. Close monitoring of patients and household contacts appears an effective measure to reduce the transmission of leprosy in endemic areas.