First report of Colletotrichum chlorophyti causing soybean anthracnose in Brazil.

Soybean [Glycine max (L.) Merr.] is one of the world's five major food crops, and Brazil produces the highest share at around 42%. Anthracnose caused by Colletotrichum is an important limiting factor to soybean production. In November 2013, anthracnose symptoms, characterized by brown irregular-shaped lesions on petioles, stems, and pods were observed in soybean fields (1% of incidence) in Vera, Mato Grosso State, Brazil. From the five plants gathered in the field, three leaves along with their corresponding petioles were meticulously chosen for the removal of symptomatic tissues. Sampling of these tissues involved carefully cutting a 0.5 × 0.5 cm fragment in the lesion area. The fragments were disinfected with 70% ethanol for 1 min, followed by 1% sodium hypochlorite for 2 min. Then the fragments were rinsed three times in sterile distilled water, placed on water-agar, and incubated at 25 °C for four days, in a 12/12 h photoperiod. Hyphal tips were transferred to potato dextrose agar (PDA) plates and incubated as previously described for seven days. A Colletotrichum sp. single-spore isolate (LFN0461) was selected, grown, preserved in filter paper, and stored at -80 °C. In 2023, it was reactivated for molecular characterization. On PDA, colony showed a rough-like mycelial growth, violaceous-black (front/reverse), with curved-shaped conidia 14.7 - 28.2 × 2.1 - 8.96 µm (average 18.4 × 4.7 µm). The DNA was extracted from 10-day-old mycelium using the cetyltrimethylammonium bromide (CTAB) method. The rDNA internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone (HIS3), and ß-tubulin 2 (TUB2) regions were amplified by polymerase chain reaction (PCR), using the primer pairs ITS-1F + ITS-4 (Gardes and Bruns 1993; White et al. 1990), GDF1 + GDR1 (Guerber et al. 2003), CYLH3F + CYLH3R (Crous et al. 2006), and Bt2A + Bt2B (Glass and Donaldson 1995), respectively. The sequences were deposited in the GenBank database (accession numbers: PP209207 - ITS; PP213392 - GAPDH; PP213393 - HIS3; MN688797 - TUB2). The reconstruction of the multilocus phylogenetic tree revealed that the LFN0461 isolate clustered with C. cholorophyti reference strain (IMI 103806) with 99.9% of Bayesian probability. Given the seed-borne nature of soybean anthracnose (Boufleur et al. 2021; Yang et al. 2013), pathogenicity tests were carried out by soybean seeds inoculation. Fifty seeds of NS6220 IPRO (Nidera) cultivar were inoculated by water restriction method, with LFN0461 colonies grown on PDA amended with mannitol (Machado et al. 2004), while 50 seeds were placed on PDA amended with mannitol as negative control. Soybean seeds remained in contact with the inoculum for 48 hours. Subsequently, seeds were sown in 2 L pots (n = 10) containing sterilized substrate, which were placed in a greenhouse at 25 ± 5 ºC. After 10 days, inoculated soybean seedlings exhibited characteristic necrotic lesions on cotyledons and hypocotyls, while negative control plants remained asymptomatic. Colletotrichum chlorophyti was successfully reisolated from the symptomatic tissues. Currently, C. chlorophyti has been reported to cause soybean anthracnose and infect seeds in the United States (Yang et al. 2013, 2012). Although this pathogen has not been reported since our first observation in 2013 in Brazil, many Colletotrichum isolates are misidentified due to reliance on morphology (Boufleur et al. 2021). To our knowledge, this study is the first report of C. chlorophyti causing soybean anthracnose in Brazil, joining a new group of emergent Colletotrichum spp. associated with this disease.

First Report of Mulberry Rust Caused by Cerotelium fici on Morus nigra in Brazil.

Leaf rust caused by Cerotelium fici (Cast.) Arth. is the main disease affecting Moraceae family plants, such as Ficus and Morus species (Galleti and Rezende 2016; Srikantaswamy et al. 2006). In August 2020, rust symptoms were observed in 100% of mulberry (Morus nigra L.) trees in an experimental orchard (Piracicaba, SP, Brazil; 22°42'28"S, 47°37'42"W). Mulberry leaves with high rust severity became yellowish and fell-off prematurely. Pustules were light brown with yellowish halo and presented mean size of 0.9 mm2. Uredinial paraphyses (n = 50) measured 42.2 ± 0.67 µm long with wall uniformly ca 0.6-1.1 µm thick. Urediniospores were brownish, echinulate, globoid to broadly ellipsoid, and measured 27.1 ± 0.29 × 21.0 ± 0.27 µm with a wall thickness of 0.6 ± 0.01 µm (n = 100). The morphology of the urediniospores observed in this study was similar to that reported in the literature for C. fici on Morus alba and Ficus spp. (Gupta et al. 1994; McKenzie 1986; Hennen et al. 2005). We used a low-coverage genome-skimming approach to retrieve genetic information of the rRNA cluster and the mtDNA. Genomic DNA was extracted from 3-4 mg of stored urediniospores at -80 °C, macerated in liquid nitrogen, using a modified cetyl trimethylammonium bromide extraction procedure (Lo Piccolo et al. 2012), and sequenced with 150-bp paired-end reads on Illumina NovaSeq 6000 System. Raw data, (45,761,957 X 2 reads) were assembled with SPAdes v3.15.1 (Bankevich et al., 2012) and the output used to create a custom BLAST database. Loci used for the phylogenetic analyses were identified by BLASTn using, as a query, sequences of C. fici from Ficus sp. from Australia publicly available: Accession No. MH047210.1 for the rRNA and MW036502.1 for COX3. The retrieved sequences were deposited in GenBank under accession numbers OM296992 and OP797407 for the partial rRNA cluster and COX3, respectively. The Bayesian inference phylogenetic analysis of the three concatenate loci (18S, 28S, and COX3) revealed that the isolate obtained in this study (MN1) was clustered in a well-supported clade with C. fici type species. Pathogenicity tests were conducted using mulberry potted plants under greenhouse conditions (25 ± 5 °C). The urediniospores suspension (5 × 104 urediniospores ml-1) with 0.05% Tween 20 was sprayed with an airbrush on fully expanded leaves until run-off. As a control, mulberry plants were sprayed with distilled water and kept under the same conditions. Inoculated and mock-inoculated plants were kept in a dark moist chamber at 23 °C (± 2 °C) for 24 h. After this period, plants were moved to the greenhouse. The experimental design was completely randomized with five replicates, each replicate consisted of one potted plant and the experiment was performed twice. At 12 days post-inoculation, all inoculated plants showed rust symptoms identical to those observed in the field, whereas control plants had no symptoms. The first symptoms were small pustules on the abaxial surface of fully expanded leaves. Small chlorotic lesions were observed on the adaxial leaf surface, which evolved into necrotic lesions. The pathogen was re-inoculated into potted plants, where it was maintained through monthly inoculations. To our knowledge, this is the first report of mulberry rust on M. nigra in Brazil. As mulberry leaves are the only natural food for silkworm (Bombyx mori L.), rust poses a significant threat to the sericulture industry because the disease can decrease production and quality of mulberry foliage.

Monocyclic Components and Photosynthetic Damage Caused by Myrtle Rust in Guava Leaves.

Austropuccinia psidii, the causal agent of myrtle rust, was, for many years, restricted to the Americas, but since reaching Hawaii in 2005, the pathogen has expanded its global range exponentially. In Brazil, myrtle rust is the main fungal disease in guava plants. Despite this, there are few studies on guava rust epidemiology. The objectives of this study were to quantify the monocyclic components of rust and to evaluate the photosynthetic damage caused by A. psidii in young and old leaves of 'Paluma' guava. The monocyclic components of guava rust and gas exchange in healthy or inoculated (105 ml-1 urediniospores of A. psidii) leaves were quantified over time. Additionally, young leaves were inoculated with varying concentrations of A. psidii inoculum, and leaf gas exchange and chlorophyll fluorescence were measured at 25 days postinoculation. The relationship between the relative CO2 assimilation of a diseased leaf (Px) and a healthy leaf (Po) is related to disease severity (x) by Px/Po = (1 - x)ß. The density of lesions, disease severity, and urediniospore production were high in young leaves, averaging 58 lesions cm-2, 50% leaf area diseased, and 2.5 × 104 urediniospores per lesion, respectively. Rust symptoms were not observed in old leaves, and resistance to infection did not cause any photosynthetic cost to these leaves. On young leaves, ß was 2.13, indicating a reduction on CO2 assimilation at green tissues from symptomatic leaves. Our data revealed that photosynthesis reduction in diseased guava leaves was caused by biochemical and photochemical damage rather than by stomatal limitation.

Anthelmintic activity of plant aqueous extracts against Panagrellus redivivus in vitro / Atividade anti-helmíntica de extratos aquosos de plantas contra Panagrellus redivivis in vitro

Control of phytonematodes is very hard and requires a combination of techniques to succeed. Alternative control through plant extracts may result in the discovery of nematicide substances. Research aimed at evaluating the effect of 33 plants submitted to aqueous extraction against Panagrellus redivivus in vitro. Concentrations were prepared at 1.25, 2.5, 5, 10, and 20%. Monitoring happened at 0, 6, 12, 24 and 30 hours after preparation. Counting considered dead nematodes subtracted from alive ones. Juveniles were also counted, and extract efficiency was expressed in percentage of control or stimuli. Data were submitted to variance analysis. Significant results got with the Scott-Knott test (5%), and multiple linear regression analysis. Extracts were observed acting as controllers, but also as stimulators to nematode reproduction. The best controlling performance was set by Carica papaya (-66% at 20%; -33.7% at 10%), Euphorbia milii (-37% at 20%), Psychotria carthagenensis (-25.5% at 2.5%), Clusia variegate (-22% at 20%), and Zamioculcas zamiifolia (-21.5% at 20%). Stimulator extracts were Mentha villosa at 10% (+148%) and 2.5% (+131.5%), followed by Aloe vera (+123% at 5%), Schinus molle (+112.5% at 10%), Schefflera arboricola (+93.5% at 5%), C. variegate (+89% at 5%), and S. molle (+88% at 5%). Some extracts kept population stable throughout the experiment, presenting lower control indexes. Besides an additive effect, there was an individual influence of concentration or time on control.(AU)

O controle de fitonematoides é muito difícil e requer uma combinação de técnicas para ter sucesso. O controle alternativo via extrato vegetal pode resultar na descoberta de substâncias nematicidas. Esta pesquisa objetivou avaliar o efeito de 33 plantas submetidas à extração aquosa contra Panagrellus redivivus in vitro. As concentrações foram preparadas a 1,25; 2,5; 5; 10; e 20%. O monitoramento ocorreu em 0, 6, 12, 24 e 30 horas após a preparação. Para a contagem, foram considerados nematoides mortos subtraídos dos vivos. Nematoides jovens também foram contados, e a eficiência dos extratos foi expressa em porcentagem de controle ou de estímulo. Os dados foram submetidos à análise de variância. Resultados significativos foram analisados pelos testes de Scott-Knott (5%) e análise de regressão múltipla. Foram observados extratos agindo como controladores, bem como estimuladores da reprodução de nematoides. A melhor performance de controle foi obtida por Carica papaya (-66% a 20%; -33,7% a 10%), Euphorbia milii (-37% a 20%), Psychotria carthagenensis (-25,5% a 2,5%), Clusia variegate (-22 a 20%) e Zamioculcas zamiifolia (-21,5% a 20%). Os extratos estimuladores foram Mentha villosa a 10% (+148%) e 2,5% (+131,5%), seguido por Aloe vera (+123% a 5%), Schinus molle (+112.5% a 10%), Schefflera arboricola (+93.5% a 5%), C. variegate (+89% a 5%) e S. molle (+88% a 5%). Alguns extratos mantiveram a população estável durante todo o experimento, apresentando menores índices de controle. Além do efeito aditivo houve uma influência individual da concentração e do tempo no controle.(AU)