Mayra Juline Gonçalves

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), β-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.

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), β-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).

The state of Santa Catarina is the second-largest producer of rice seeds in Brazil. Research on phytopathogenicbacterias in this crop is scarce and the high frequency of panicle diseases leads to the hypothesis that seeds may be infected by bacteria. This research quantified the incidence of bacteria in the seeds, verified the bacteria viability during the storage period and characterized the associated bacteria. Seeds from the 2018/19 and 2019/20 seasons were analyzed. To check the incidence, the seeds were disinfected, plated on a nutrient agar + fungicide culture medium, and incubated for seven days at 27 °C. To assess viability, every 45 days, three cultivars stored in a processing unit were subjected to the same detection methodology. To characterize, prevalent colonies were isolated on semi-selective culture medium Pantoea genus-specific agar (PGSA), where the ones that showed growth were subjected to deoxyribonucleic acid (DNA) extraction and Polymerase Chain Reaction (PCR), DNA sequencing, and sequence comparison on GenBank. The hypersensitivity reaction (HR) in tobacco was performed using a bacterial suspension of each isolate. All seed samples had an average incidence of 83%. During storage, the seeds maintained stable bacterial viability, with an average incidence of 95% at the beginning of storage and 99% at the end of it. All isolates that grew in PGSA culture medium were identified by molecular characterization with 100% identity with two specimens of Pantoeaananatis and one of them induced RH in tobacco.

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.

M. phaseolina is a cosmopolitan pathogen with a wide range of hosts and over 500 different species, including economic crops, such as bean (P. vulgaris L.), maize (Zea mays L.), soybean (Glycine max (L.) Merrill), sorghum (Sorghum bicolor (L.) Moench, peanut (Arachis hypogaea L.), sunflower (Helianthus annuus L.) and cotton (Gossypium hirsutum L.) (⁷, ¹¹, ¹³, ¹⁸).

Gibberella ear rot (GER) in maize, caused by Fusarium graminearum species complex, is a destructive disease in southern Brazil and worldwide. Five field-experiments were conducted to determine fungicide efficacy, grain yield and grain quality (study I), as well as response of increasing rates (study II) on GER control, grain yield (GY) and grain damage (GD) in maize during the 2014/15 and 2015/16 growing seasons. Fungicides were sprayed 48 h before (preventive action) or 48 h after (curative action) inoculation of two Fusarium meridionale isolates (PR and MG). Study I consisted in applying nine active ingredients (metconazole, pyraclostrobin, tebuconazole, trifloxystrobin, prothioconazole, thiophanate-methyl, azoxystrobin, cyproconazole and carbendazim) present in eight commercial formulations at two sowing dates. Study II consisted in applying increasing rates of three fungicides evaluated at three sowing dates using only PR isolate. In study I, no differences were observed among fungicides when the variables GER, GY and GD were analyzed together. There were interactions between time of fungicide application and F. meridionale isolates when GER severity and GY were considered. Preventive fungicide application at different sowing dates and the use of MG isolate reduced GER by 11.6% and increased GY by 5.3%, compared to curative application. In study II, there was a significant interaction between fungicides and application time for GER, GY and GD. Preventive application of trifloxystrobin + prothioconazole (0.15 + 0.17 and 0.22 + 0.26 L ha^-1), pyraclostrobin + metconazole (0.19 + 0.12 and 0.29 + 0.18 L ha^-1) and carbendazim (2 L ha^-1) reduced GER by 41 and 50%, 47 and 54%, and 54% at the three sowing dates, respectively. There was an increment of 45.7% and 46.8% in GY when trifloxystrobin + prothioconazole (0.15 + 0.17 and 0.22 + 0.26 L ha^-1), respectively, were preventively sprayed at the three sowing dates, compared to the control treatment. Trifloxystrobin + prothiocanazole, pyraclostrobin + metconazole and carbendazim (1.0 and 2.0 L ha^-1) preventively sprayed at two rates reduced GD incidence by 53.5 and 48.7%, 47.7 and 57.5%, 48.1 and 50.5%, respectively, for each fungicide and rate, and could be incorporated into a disease management program.

No-till system and high plant density are common practices in soybean crops of the Brazilian southern region, which has increased the incidence of stem and pod diseases. The objective of this study was to evaluate the effect of plant populations on stem blight incidence. The experiments were conducted in Muitos Capões Municipality, Rio Grande do Sul State, during the seasons 2006/07 and 2007/08, using cultivars BRS 255 RR and CD 213 RR. The evaluated populations had 12, 18, 24, 30 and 36 plants m^-2, spaced 0.45 m between rows. The experimental plot units consisted of 5m width and 10m length, arranged in randomized blocks with four replicates. Stem blight incidence was quantified at phenological stages R5.1, R5.5, R6 and R7 and the collected data underwent regression analysis. Higher soybean plant densities caused a positive and significant increase on stem blight incidence in all four phenological stages for both cultivars and seasons. There was no significant difference for soybean grain yield according to the increase in plant population. Results indicated that soybean management preventing high plant densities can contribute to reduce stem blight incidence.

The Bemisia tabaci is a polyphagous insect and a successful vector of plant viruses. B. tabaci is a species complex and in Brazil native species from the New World (NW) group, as well as the invasive species, Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED) were reported. For better understanding the distribution of the different species four years after the Mediterranean species invasion in Brazil, whiteflies were collected from 237 locations throughout the country between the years of 2013 and 2017, species were identified and the facultative endosymbionts detected. The survey revealed that MEAM1 was the prevalent species found on major crops across Brazil. It is the only species present in North, Northwestern and Central Brazil and was associated with virus-infected plants. MED was found in five States from Southeast to South regions, infesting mainly ornamental plants and was not associated with virus-infected plants. The prevalent endosymbionts identified in MEAM1 were Hamiltonella and Rickettsia; and the mtCOI analysis revealed low genetic diversity for MEAM1. In contrast, several different endosymbionts were identified in MED including Hamiltonella, Rickettsia, Wolbachia and Arsenophonus; and two distinct genetic groups were found based on the mtCOI analysis. Monitoring the distribution of the whiteflies species in Brazil is essential for proper management of this pest.

Biodiversidade de basidiomicetos encontrados em um fragmento de Floresta Ombrófila Mista Basidiomycetes biodiversity founded in a fragment of Araucaria Forest