First Report of Seed Decay Caused by Diaporthe ueckeri on soybean in Brazil.

A survey of Diaporthe/Phomopsis Complex (DPC) species was carried out on 479 asymptomatic soybean (Glycine max (L.) Merrill) seed samples collected from commercial soybean fields in the states of Santa Catarina (20 counties) and Rio Grande do Sul (41 counties), in the 2020/21 (n=186), 2021/22 (n=138) and 2022/23 (n=155) seasons from 120 cultivars. The seeds were provided by seed producers who collected according to the sampling standard of the Ministry of Agriculture, Livestock and Food Supply. From each sample received, 200 symptomless seeds were randomly sorted out. The seeds were surface disinfected by immersion in a sodium hypochlorite solution (1%) for two minutes and placed on Potato Dextrose Agar (PDA). The plates were incubated for 7 days at 23°C with a photoperiod of 12-h. The average prevalence of 73.7% of DPC-infected seeds. Colonies were isolated by transferring mycelial tips to PDA and incubating for 14 days at 25ºC in a 12-h photoperiod. One colony (isolate MEMR0500) had morphological characteristics similar to those reported in Lopez-Cardona (2021). This isolate had a floccose, dense colony ranging from grayish beige to brown with greenish regions and black globose pycnidia (3 to 4 pycnidia/cm²). Alpha-conidia, 5.1 to 7.0 µm x 1.5 to 2.8 µm, were observed after 30 days and were hyaline, aseptate and fusiform (Figure S1). No beta-conidia were observed. Soybean plants of cultivars BMX Cromo IPRO, BMX Zeus IPRO, BRS 5804 RR, FPS 1867 IPRO and NEO 750 IPRO were tested for pathogenicity using the toothpick inoculation method (Siviero and Menten 1995). Non-colonized toothpicks served as a negative control. Plants were incubated for four days at 25°C and 90% relative humidity. Elongated 1.0 to 2.5 cm x 0.5 to 0.9 cm lesions gray-brown/reddish-brown with a depressed center were observed in all inoculated cultivars. The fungus was reisolated and the characteristics of the colonies were identical to those previously isolated. For molecular characterization, DNA was extracted from the mycelia using the CTAB method (Doyle and Doyle 1990). End-point PCR was performed using GoTaq® Flexi DNA Polymerase (Promega, USA) and primer pairs, ITS-4F/ITS-5, T2/Bt2b and EF1-728F/EF1-986R to amplify the internal transcribed spacer (ITS) (Costamilan et al. 2008), ß-tubulin (TUB2) (Glass and Donaldson 1995), and translation elongation factor 1-α (TEF1) (Carbone and Kohn 1999) genes, respectively. The amplified fragments were sequenced and submitted to blast search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) with the sequences available in GenBank. The fragment from ITS (accession number OR912979) showed 99.8% (549/582 bp) identity with Diaporthe ueckeri Udayanga & Castl. [as 'ueckerae'] [syn. D. miriciae R.G. Shivas, S.M. Thomps. & Y.P. Tan] isolate FAU656 (Ac. N. KJ590726). The sequence of TEF (Ac. N. PP372869) showed 99.7% (339/355 bp) identity with D. ueckeri FAU656 (Ac. N. KJ590747), and of TUB (Ac. N. PP372870) showed 98.9% (436/536 bp) identity with D. ueckeri FAU656 (Ac. N. KJ610881). A phylogenetic tree with amplified sequences of each gene and the corresponding representative sequences from the DPC was constructed in MEGA X (Kumar et al. 2018). The MEMR0500 isolate was clustered only with the D. ueckeri clade, confirming the identity of the fungus (Figure S2). In Brazil, this is the first report of the association of this pathogen with soybean seeds. In other countries, this pathogen has been identified as the causal agent of stem canker (Mena et al. 2020; Lopez-Cardona et al. 2021). Further research is needed to analyze the risk of this seed-associated pathogen.

First Report of Colletotrichum fructicola Causing Anthracnosis on Glycine max in Brazil.

Brazil is the largest producer of soybean [Glycine max (L.) Merrill], cultivated in diverse environments and systems. This scenario can contribute to emergence of new diseases or increase the severity of secondary diseases. In March 2023, elliptical to circular, brownish lesions, 5.2-6.1 cm length and 1.1-1.5 cm width, with salmon-colored masses of conidia in the center of the lesions, were observed on the stems of soybean cultivar 'CZ 16B17 IPRO', in the municipality of Campos Novos, Santa Catarina, Brazil (27º25'19''S and 51º14'14''05W). The presence of 210-355 µm length and 210-232 µm width acervuli was rare, with arrows larger than the mass of conidia (Figure S1). Fragments of the infected tissues were cut, disinfected and placed in Petri dishes containing Potato Dextrose Agar (PDA) or V8-agar medium and maintained at 23 ± 2ºC and a photoperiod of 12 h dark-light cycle. After 13 days, the development of grayish-white colonies was observed on both culture media, with the formation of a mass of septate hyaline, oblong, cylindrical conidia, 13.3-15.3 µm length and 2.9-3.5 µm width, with obtuse ends. One pure monosporic isolate was selected, isolate CF1. The presence of sexual structures was observed on PDA after 13 days and in V8 after 15-20 days. Perithecia were dark brown and globose, either immersed in the culture medium or on the surface between the mycelia. Inside of perithecia, unitunicate, clavate, and cymbiform asci, 39.1-61.0 µm length and 9.6-11.7 µm width were observed, containing eight spindle-shaped and slightly curved ascospores with rounded tips 13.8-18.3 µm length and 3.0-4.2 µm width (Figure S1). Pathogenicity tests were performed on young soybean plants at V1 phenological growth stage in four repetitions. PDA disks, 7mm in diameter, with growth mycelium were placed on stems while using uninfected PDA disks as a control. Plants were incubated in a chamber at 25 ± 2°C and 90% relative humidity. Anthracnose lesions were observed only on the stems of the inoculated plants. The same pattern of symptoms was observed on the stems, and the fungus were reisolated on PDA. The colony and morphological characteristics were identical to the previously isolated fungus. For molecular characterization, the growth mycelia were collected, macerated in liquid nitrogen, and DNA was extracted using the method Doyle and Doyle (1990) with CTAB. End-point PCR was performed using the GoTaq® Flexi DNA Polymerase (Promega, USA) and the primers, ITS-1F/ITS-4, T1/Bt2b, CL1C/CL2C, GDF/GDR, and SODglo2-F/SODglo2-R (Weir et al. 2012) for the amplification of internal transcribed spacer (ITS), ß-tubulin (TUB2), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and superoxide dismutase (SOD), respectively. Amplified fragments were sequenced and compared with the available sequences in the Genbank (www.ncbi.nlm.nih.gov/genbank/). The sequences of all five-genes (Accession numbers OR883777, OR891749, OR891750, OR891751 and OR891752, respectively) of the isolate CF1 characterized in this study showed 99% nucleotide identities whith the stand isolate ICMP 18581 of Colletotrichum fructicola. A phylogenetic tree was constructed in MEGA X (Kumar et al. 2021), containing the amplified and concatenated sequences and representative species from the Colletotrichum gloeosporioides complex. The isolate grouped only with C. fructicola clade, confirming the identity of the fungus (Figure S2). To our knowledge, this is the first study reporting the infection of C. fructicola in soybeans in Brazil, which has already been reported in China (Xu et al. 2023).

First report of Leek yellow stripe virus, Onion yellow dwarf virus, and the putative allium polerovirus A in elephant garlic (Allium ampeloprasum) in Brazil.

Five elephant garlic plants (Allium ampeloprasum L.) showing leaf symptoms of chlorotic streaks and mosaic (Figure 1A and B) were collected, in September 2021, in an experimental area in municipality of Rio do Sul (27°11'07"S, 49°39'39"W), State of Santa Catarina, Brazil. Total RNA was extracted using TRIzol® reagent (Invitrogen, USA), according to the manufacturer's instructions to investigate viral infection. The RNA from all five plants were pooled into a single sample for cDNA library construction with the TruSeq Stranded Total RNA with Ribo-Zero Plant (Illumina) kit, which was then sequenced on the Illumina HiSeq2500 platform (Proteimax Biotechnology LTDA). After high throughput sequencing (HTS), 49 million raw reads (each 151nt) were generated. They were trimmed with the BBduk tool and de novo assembled with the Tadpole assembler tool (Geneious Software version 2022). A total of 28,345 contigs were generated and searched against the NCBI virus genome database using BLASTn and BLASTx, with positive results for two potyviruses, leek yellow stripe virus (LYSV), onion yellow dwarf virus (OYDV), and the putative polerovirus allium polerovirus A (APVA). The trimmed reads were mapped with the BBmap tool (Bushell 2014), using reference sequences for LYSV (NC_004011), OYDV (NC_005029), and APVA isolate Won (MH898527). A total of 806,060 reads were mapped, resulting in the nearly complete genome of LYSV (isolate RDS22-2, 10,268 bp, ON565071), which shared the highest (89.41%) nucleotide (nt) identity with LYSV isolate MG (KP258216). The nearly complete genome of OYDV (isolate RDS22-1, 10,519 bp, ON565070) was assembled using 311,467 reads, being 90.21% nt identical to OYDV isolate G-118 (KF632714). The APVA genome (isolate RDS22-3, 4,367 bp, ON565072, Figure 1C) was assembled from 116,303 reads and it shared the highest (90.73%) nt identity with APVA isolate Won. Subsequently, each sample was RT-PCR screened separately for potyviruses and poleroviruses, using the generic primer pairs NIb2F/NIb3R (Zheng et al., 2010) and Pol-G-F/Pol-G-R (Knierim et al., 2010), respectively. Amplified DNA fragments with approximately 350 bp and 1000 bp were obtained for potyviruses and poleroviruses, respectively, and were sent for Sanger sequencing (ACTGene, Alvorada, Brazil). The Sanger derived partial sequences shared 98 to 100% nt identities with corresponding HTS-derived sequences. The most common virus was LYSV, which was found in three of the five tested samples, whereas OYDV and APVA were only found in one sample each. The plants were also screened with specific primers for each virus, and none of the samples revealed mixed infections. Elephant garlic is primarily utilized for industrial garlic production in several countries, and it is now being researched in Brazil for the same purpose. It can be observed from this study that elephant garlic is susceptible to two of the most common viruses in garlic (LYSV and OYDV), which must be considered in the future while developing resistant varieties or in using thermotherapy and shoot tip/meristem culture to recover virus-free cultivars. LYSV and OYDV have already been described in Brazil infecting Allium sativum (Kitajima 2020). The only complete APVA sequence available is from China (Isolate Won), but no further characterization of the virus has been performed and published. The occurrence of this virus in Brazil highlights the importance of further research to obtain a more robust virus characterization.

First report of maize rayado fino virus in corn fields in Santa Catarina, Brazil.

Maize (Zea mays L.) is the main cereal food of humans and animals in Brazil. In 2020 and 2021, a severe infestation of corn leafhoppers (Dalbulus maidis; Hemiptera: Cicadellidae) was observed in Santa Catarina State (South of Brazil). Subsequently, symptoms of chlorotic stripes limited in leaf veins started to appear in maize plants. Given the similarity of symptoms and the presence of high populations of corn leafhoppers in corn production areas, 30 plants in reproductive stage showing systemic symptoms were collected in summer and autumn from commercial fields of five municipalities in Santa Catarina: Campos Novos (27°23'18.0"S, 51°12'52.7"W), Lages (27°47'17.8"S, 50°18'16.9"W), Mafra (26°06'42"S, 49°48'25"W), Fraiburgo (27°01'36"S, 50°55'19"W), and Abelardo Luz (26°34'02"S, 52°20'02"W). The young leaves of these samples were used for molecular analyses targeting the maize rayado fino virus (MRFV; Tymoviridae: Marafivirus). Total nucleic acids were extracted using TRIzol® (Invitrogen, USA), following the manufacturer's instructions. These were used as a template for cDNA synthesis with the enzyme MMLV-RT (Promega, USA), following the manufacturer's instructions. The polymerase chain reaction (PCR) was performed using Gotaq® DNA polymerase (Promega, USA) and MRFV-09/MRFV-10 primers (Hammond et al. 1997). All PCR products were subjected to electrophoresis in 1% agarose gel and were visualized under ultraviolet light. Twenty-eight of the 30 tested plants were MRFV-positive, showing a fragment with an expected size of ~633 bp. To confirm our results, all MRFV-positive samples were sent for sequencing (GenBank accession numbers OM763708 - OM763710 and ON730784 - ON730806) and submitted to BLASTn search (https://blast.ncbi.nlm.nih.gov/Blast.cgi), resulting in identities ranging from 96.21% to 99.21% with the isolate "Brazil 26" of MRFV, which was detected in 2005 in São Paulo, Brazil (GenBank accession nº: AF186178) (Hammond and Bedendo 2005). A second set of primers was used to validate the first PCR, confirming MRFV infection (data not shown).Moreover, whitish streaks and leaf reddening were observed on the leaves of some plants; therefore, the identification for phytoplasmas (Candidatus Phytoplasma asteris) and spiroplasmas (Spiroplasma kunkelii) from the corn stunt complex was performed. For this, previously extracted nucleic acids from each sample were used as templates for a multiplex PCR using the primers CSSR6/CSSF2 and R16F2n/R16R2 (Gundersen and Lee 1996; Barros et al. 2001). Two plants were infected with only spiroplasma, 17 samples were infected with Spiroplasma and MRFV, and three samples were infected by these three pathogens. An increasing incidence of corn stunt has been observed in commercial fields in Santa Catarina in recent years. Mollicutes are commonly found and mostly studied as causal agents of corn stunt disease. On the contrary, despite being present in Brazil since the 1970s, the virus is less studied because its contribution to the corn stunt complex is still unknown (Hammond and Bedendo 2001). In this report, indications that the virus is expanding to different regions in southern Brazil were observed, which raises an opportunity for further evaluation and its consideration in monitoring programs. Moreover, to the best of our knowledge, this is the first report of MRFV in Santa Catarina, Brazil.

First report of Podosphaera macularis causing hop (Humulus lupulus) powdery mildew in Brazil.

The hop (Humulus lupulus L.) is a dioecious perennial climbing plant grown commercially worldwide. Wild hops are widely distributed throughout the Northern Hemisphere, Europe, Asia, and North America (Neve, 1991). In the Southern Hemisphere, some of the leading hop-producing countries include South Africa, Australia, and New Zealand. Brazil began hop production less than 5 years ago. In January 2019, amphigenous white powdery circular fungal colonies were observed on the leaves and stems of hop plants (cultivar Chinook) within a 900m2 hop garden in Lages municipality, Santa Catarina State, southern Brazil. The incidence of the disease was present on almost 100 per cent of "Chinook" cultivar plants and diseased foliage was collected to identify the pathogen and used to inoculate healthy plants. Hop powdery mildew lesions with hyaline and septate mycelium with chains of unicellular conidia (n =100) hyaline, barrel-shaped, mean of length/width ± standard deviation 25-27 × 13-18 µm ± 0.980, with fibrosin bodies, and conidiophores erect with cylindrical foot cells, were visible within 10 days. The causal agent was identified as Podosphaera macularis (Wallr.:Fr.) Lind (synonym S. humuli (DC.) Burrill) on the basis of conidial shape, size and host range (Royle 1978; Braun 1987; Mahaffee et al., 2009), complemented with the present molecular analysis. Chasmothecia have not been observed in the field to date. A conidial suspension of 200 ml at concentration of 1.4 x 105 was mixed with 5ul of Tween® 20 for the pathogenicity assay. Ten plants of 9-month-old of hop "Chinook" cultivar, were inoculated with 5 ml of the conidial suspension using a manual spray. The control plot was only sprayed with water. The inoculated plants were maintained at 22ºC ± 1ºC with a 12-hour photoperiod and 65% relative humidity. White mycelia were visible first on the adaxial leaf surfaces of the inoculated younger leaves after 10 days and the disease severity reached between 2 to 5%. No symptoms were observed at the control plot. P. macularis infected most aerial plant tissues of the inoculated plants and caused approximately 50% of cones losses. P. macularis conidia were collected from the infected leaf tissue with a sterile soft camel-hair brush and DNA was extracted using a Wizard Genomic DNA extraction kit. The primers ITS1/ITS4 (White et al., 1990) were used to amplified and sequenced a fragment of the ITS region. PCR products were subjected to Sanger Sequencing to confirm sample species. The resulting 522-bp sequence was deposited into GenBank (accession n°. MN630490). BLASTn showed a 99.81% sequence identity with the CT1 isolate of P. macularis from H. lupulus (MH687414). The presence and identification of P. macularis in hop production regions is a new challenge to growers in Brazil. Research related to the knowledge of the disease cycle, epidemiology, and control strategies for the integrated management should be conducted, as there are no registered fungicides for powdery mildew on hop in Brazil. To our knowledge, this is the first report of P. macularis in Brazil, as well as in South America. References Braun, U. (1987) A Monograph of the Erysiphales (Powdery Mildews). J. Cramer, Berlin, German Democratic Republic. p 113. Mahaffee, W. F., Pethybridge, S.J., Gent, D.H (2009) Compendium of hop diseases and pests. The American Phytopathological Society Press, Saint Paul, Minnesota. Neve R. A (1991). Hops. Chapman and Hall: London. Royle, D. J (1978). Powdery mildew of the hop. Pages 381-409 in: The Powdery Mildews. D. M. Spencer, ed. Academic Press, New York. White, T. J., Bruns, T., Lee, S., and Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. pp. 315-322 in: PCR Protocols: A Guide to Methods and Applications. M. Innis, D. Gelfand, J. Sninsky, and T. White, eds. Academic Press, San Diego.

Validation and optimization of the loop-mediated isothermal amplification (LAMP) technique for rapid detection of wheat stripe mosaic virus, a wheat-infecting pathogen.

BACKGROUND: Wheat stripe mosaic virus (WhSMV) is a significant wheat pathogen that causes substantial yield losses in Brazil and other countries. Although several detection methods are available, reliable and efficient tools for on-site WhSMV detection are currently lacking. In this study, a Loop-Mediated Isothermal Amplification (LAMP) method was developed for rapid and reliable field detection of WhSMV. We designed WhSMV-specific primers for the LAMP assay and optimized reaction conditions for increased sensitivity and specificity using infected plant samples. RESULTS: We have developed a diagnostic method utilizing the Loop-Mediated Isothermal Amplification (LAMP) technique capable of rapidly and reliably detecting WhSMV. The LAMP assay has been optimized to enhance sensitivity, specificity, and cost-effectiveness. CONCLUSION: The LAMP assay described here represents a valuable tool for early WhSMV detection, serving to mitigate the adverse economic and social impacts of this viral pathogen. By enabling swift and accurate identification, this assay can significantly improve the sustainability of cereal production systems, safeguarding crop yields against the detrimental effects of WhSMV.