ABSTRACT
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.
ABSTRACT
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).
