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Macrophomina phaseolina (Tassi) Goid. is a fungal pathogen that causes root and stem rot in several economically important crops. However, most of disease control strategies have shown limited effectiveness. Despite its impact on agriculture, molecular mechanisms involved in the interaction with host plant remains poorly understood. Nevertheless, it has been proven that fungal pathogens secrete a variety of proteins and metabolites to successfully infect their host plants. In this study, a proteomic analysis of proteins secreted by M. phaseolina in culture media supplemented with soybean leaf infusion was performed. A total of 250 proteins were identified with a predominance of hydrolytic enzymes. Plant cell wall degrading enzymes together peptidases were found, probably involved in the infection process. Predicted effector proteins were also found that could induce plant cell death or suppress plant immune response. Some of the putative effectors presented similarities to known fungal virulence factors. Expression analysis of ten selected protein-coding genes showed that these genes are induced during host tissue infection and suggested their participation in the infection process. The identification of secreted proteins of M. phaseolina could be used to improve the understanding of the biology and pathogenesis of this fungus. Although leaf infusion was able to induce changes at the proteome level, it is necessary to study the changes induced under conditions that mimic the natural infection process of the soil-borne pathogen M. phaseolina to identify virulence factors.

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Macrophomina phaseolina is a soil-borne pathogenic fungus that infects a wide range of crop species and causes severe yield losses. Although the genome of the fungus has been sequenced, the molecular basis of its virulence has not been determined. Identification of up-regulated genes during fungal infection is important to understand the mechanism involved in its virulence. To ensure reliable quantification, expression of target genes needs to be normalized on the basis of certain reference genes. However, in the case of M. phaseolina, reference genes or their expression analysis have not been reported in the literature. Therefore, the objective of this study was to evaluate 12 candidate reference genes for the expression analysis of M. phaseolina genes by applying three different fungal growth conditions: a) during root and stem infection of soybean, b) in culture media with and without soybean leaf infusion and c) by inoculating a cut-stem. Based on BestKeeper, geNorm and NormFinder algorithms, CYP1 was identified as the best recommended reference gene followed by EF1ß for expression analysis of fungal gene during soybean root infection. Besides Mp08158, CYP1 gene was found suitable when M. phaseolina was grown in potato-dextrose broth with leaf infusion. In the case of cut-stem inoculation, Mp08158 and Mp11185 genes were found to be most stable. To validate the selected reference genes, expression analysis of two cutinase genes was performed. In general, the expression patterns were similar when the target genes were normalized against most or least stable gene. However, in some cases different expression pattern can be obtained when least stable gene is used for normalization. We believe that the reference genes identified and validated in this study will be useful for gene expression analysis during host infection with M. phaseolina.

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Stevia (Stevia rebaudiana [Bertoni] Bertoni) is a perennial plant originating in Paraguay. Stevia is primarily cultivated for the production of non-caloric sweeteners. In December 2018, wilted stevia cv. 'PC4' were recovered from two separate fields of 0.3 ha (24.66 S 56.46 W) and 0.5 ha (24.69 S 56.44 W), both with 3 years history of stevia production in San Estanislao County, San Pedro, Paraguay. The wilted plants were randomly distributed in beds covered with plastic mulch and a 30% disease incidence was recorded. Dark brown septate hyphae and microsclerotia were observed on stem bases and black necrotic roots of the wilted plants. Root and crown regions were washed, cut into 0.5 to 1.0 cm pieces, and then surface-disinfested with 0.6% NaOCl before placing them in Petri dishes containing acidified potato-dextrose-agar. Plates were incubated for one week at 25 ± 5°C under fluorescent light with a 12 h photoperiod yielding five isolates SP1PY, SP2PY, SP3PY, SP4PY and SP5PY with gray-black colonies without conidia but showing numerous microsclerotia. Twenty microsclerotia from pure cultures of five isolates were measured, with mean width 38.8 ± 4.7 µm and length 68.8 ± 15.5 µm. Fungal DNA was extracted from mycelia of five isolates for PCR amplification of the internal transcribed spacer (ITS) and translation elongation factor 1-alpha (TEF1-α) using ITS4/ITS5 and EF1-728F/EF-2 primers (Machado et al. 2019). The resultant amplicons were sequenced at Eton Bioscience (Research Triangle Park, NC) and deposited in the NCBI GenBank database (ITS: MT645815, OM956150, OM956151, OM956152, OM956153; and TEF1-α: MT659121, OM959505, OM959506, OM959507, OM959508). Sequences were aligned with several isolates of Macrophomina spp. previously reported (Huda-Shakirah et al. 2019; Machado et al. 2019; Santos et al. 2020; Poudel et al. 2021) using ClustalW. Alignments (ITS and TEF-1α) were concatenated to generate a maximum likelihood tree using MEGA7. The novel isolates grouped into the M. euphorbiicola clade with 95% of bootstrap support. Stevia plants cv. 'Katupyry' were grown in 10 cm-diameter nursery bags containing autoclaved sandy soil and kept under greenhouse conditions (28 ± 5°C; 16 h photoperiod). Fifteen plants per isolate (n=75) were inoculated by adding 20 g of rice infested with M. euphorbiicola to each plant. Infested grains were distributed around the crown of the plant at a depth of 0.5 cm; non-infested rice was added to four control plants. Lower-stem lesions and microsclerotia of M. euphorbiicola developed on all inoculated plants. No lesions or microsclerotia were observed on control plants. The M. euphoribiicola fungus was re-isolated from inoculated stevia plants but not from the non-infested rice treated plants. Koch's postulates were repeated twice with similar results. Previously, M. phaseolina was reported causing charcoal rot on stevia in Egypt (Hilal and Baiuomy 2000), and in North Carolina, USA (Koehler and Shew 2017). However, Paraguayan isolates grouped with isolates of M. euphorbiicola based on the combined sequences of the ITS and TEF-1α regions. Machado et al. (2019) reported M. euphorbiicola causing charcoal rot on castor bean (Ricinus communis) and bellyache bush (Jatropha gossypifolia) in Brazil, which borders northeast Paraguay, a major stevia production area. This pathogen has a significant impact on stevia production during hot, dry weather by reducing the number of harvestable plants and increasing replanting costs in perennial production systems.

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Two new macrocyclic diterpenes, 2-epi-latazienone (4) and 15ß-acetoxy-7ß-nicotinoyloxy-3ß,8α-di-(2-methylpropanoyloxy)-4αH,9αH,l1αH-lathyra- 5E,12E-dien-14-one (5), and three known lathyrane-type diterpenes (1-3) were isolated from Euphorbia laurifolia latex. Their structures were determined on the basis of a detailed analysis of their 1D and 2D NMR spectroscopic and mass spectral data.

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