First report of Epicoccum nigrum causing leaf spots on Campomanesia guazumifolia (Camb.) Berg in Brazil.

Campomanesia guazumifolia is a native tree that produces fruit that can be consumed fresh or used by industry (Donadio et al., 2002). In February 2022, in the experimental area of the Universidade Tecnológica Federal do Paraná - Brazil, disease was observed in 22 trees, with 50% to 80% severity in crown leaves. Symptoms were small, irregular, or circular-shaped, dark-brown lesions with yellow halos (Figure S1). As the disease progressed, the lesions increased in size, without distinction between mature and young tissues, causing complete leaf wilting. Twenty symptomatic leaves from 11 trees grown in the same orchard line were collected. For fungal isolation, the leaf surfaces were disinfected with 0.5% NaOCl solution for 1 min, rinsed in sterile distilled water, and dried on sterile filter paper. Five fragments of diseased leaf tissue were placed on a potato dextrose agar medium. The morphological characteristics of the colony, such as filamentous mycelium and golden yellow on the upper part, with the presence of circular to ovoid and multicellular conidia (mean 21.00 µm x 24.45 µm, n = 30) of the nine isolates, coincided with the description of the fungus of the genus Epicoccum (Valenzuela-Lopez et al., 2018). Further identification of one of these nine isolates was confirmed by amplifying and sequencing three loci (ITS, ß-tubulin, and RPB2) using the ITS1/ITS4, Bt2a/Bt2b, and 5F2/7cR primer pairs, respectively (White et al., 1990, Glass and Donaldson, 1995, O'Donnell et al., 2007). A single representative isolate (Cgen01) was analyzed and submitted to GenBank (OR020968, OR079879, and OR079878). The Bayesian Inference was used to reconstruct the phylogenetic trees (Figure S2), starting from random trees for 5,000,000 generations, using MrBayes v. 3.2.1 (Ronquist et al., 2012). The isolate clustered together with the isolate of Epicoccum nigrum (Chen et al., 2017) with a high posterior probability (0.98). For the pathogenicity tests, four young, healthy branches containing 20 leaves were spray-inoculated with 1.5 mL of conidia suspension of Cgen01 (106 conidia mL-1), covered with perforated transparent plastic bags, and moistened with distilled water in the orchard. The air temperature ranged from 14ºC to 25ºC. Sterile distilled water was used as a control. Three replicates (pathogen and control) on different trees were evaluated. After five days, the fungus was re-isolated from the symptomatic lesion, showing morphological characteristics similar to those of Cgen01. Control branches did not show fungal growth. The inoculation test was conducted twice and similar symptoms were observed. This is the first report of leaf spots caused by E. nigrum on C. guazumifolia in Brazil. E. nigrum, an endophytic fungus described as a mycoparasite, showed phytopathogenic behavior in this study, causing spots and loss of leaves in C. guazumifolia, drastically reducing the production of photoassimilates and affecting the quality of the fruits.

First report of Myrtle Rust caused by Austropuccinia psidii on Myrcianthes pungens (O. Berg) D. Legrand in Paraná state, Brazil.

Myrcianthes pungens is a tree fruit native to Brazil, unknown to a large part of the population, with fruit consumed only locally. In October 2022, at the experimental area at Universidade Tecnológica Federal do Paraná (UTFPR) in the Dois Vizinhos city, Paraná State, Brazil, symptoms of the disease were observed on mature leaves and fruits of 17 trees. Fungal fructifications were observed in the form of bright yellow uredinia containing a large mass of urediniospores on the surface and on the leaves and fruits that resembled the structures typical of a Myrtaceae rust pathogen. Leaves colonized by the fungus showed deformations, turning dark and rapidly causing senescence. In the orchard, the fungus affected 80% of the trees, with a severity of 40 to 45%. Diseased fruits (10) and leaves (10) (from each tree) were collected from 17 trees from different positions in the orchard. The observed structures (optical microscope) were hyaline and globose urediniospores (n = 30) which had pointed echinulate ornaments throughout their surface (Cummins & Hiratsuka, 2003), (n = 30, 14.84 µm × 21.1 µm). These characteristics were similar to the morphological characteristics of the genus Austropuccinia previously described by Young et al. (2019). A strain was selected as a representative for molecular characterization and pathogenicity tests (accession no. APM001). For molecular identification, the internal transcribed spacer (ITS) region (Kroop et al., 1995), b-tubulin (TUB2), and translation elongation factor 1-alpha (TEF) (Machado et al., 2015) were amplified by PCR and sequenced. The sequences were deposited in GenBank (accession nos. ITS: OQ442638, TUB2: OQ506543, and TEF: OQ506542). Phylogenetic analyses using Bayesian inference grouped the isolate with the type species of Austropuccinia psidii with a high posterior probability (1.0). Pathogenicity tests used conidial suspensions (1x105urediniospores/ml). Four branches containing twenty leaves and two young asymptomatic fruits were individually inoculated with 1.5 mL of urediniospore suspension using a bottle with a spray nozzle cap. The branches were protected with perforated transparent plastic bags moistened with distilled water and incubated at room temperature (18 ºC to 25 ºC). Three replicates (pathogen and control) spread on different trees in the orchard were used in this experiment. After seven days, symptoms of rust appeared on the leaves and on the tenth day of the fruits, with morphological characteristics similar to those previously reported. Control branches showed no fungal growth. The inoculation test was repeated, confirming the symptoms. This is the first report of the incidence of rust caused by A. psidii on leaves and fruits of M. pungens in Paraná State. The importance of the disease is due to the high percentage of fruit loss due to rapid rot and drop caused by the pathogen attack.

First Report of Myrtle Rust Caused by Austropuccinia psidii in Campomanesia guazumifolia (Camb.) Berg in Brazil.

Campomanesia guazumifolia is a Brazilian fruit tree that has ecological importance and the potential to be explored by the food and medical industries (Lima et al., 2011). In February 2019, in the experimental orchard at the Universidade Tecnológica Federal do Paraná, Dois Vizinhos city, Paraná State - Brazil, disease symptoms were observed on leaves, stems, and fruits of 22 C. guazumifolia trees. Yellow uredinia were observed on upper side of the leaves, stems, and flowers, which resembled typical uredinia of Myrtaceae rust. The pustules occurred mainly on young shoots, and on flowers, they infected their sepals. Over time, tissues colonized by the pathogen exhibited deformations and mummification occurred in infected fruits. In the orchard, the fungus affected 80% yield. Twenty diseased plant parts (from each of the eleven trees) were collected at different positions in the orchard. One strain were selected as a representative for morphological characterization, multilocus phylogenetic analysis, and pathogenicity tests. The structures observed were epiphyllous uredinia (leaves), united in small groups with hyaline and obovoid or obpyriform urediniospores, which presented echinulate ornaments, germinated pores in the subequatorial and inordinate positions (Cummins; Hiratsuka, 2003) (n = 30, 14.84 x 21.12 µm). The morphology of uredinia and urediniospores was similar to the morphological characteristics of the genus Austropuccinia previously described in Young (2019). For molecular identification, genomic DNA was extracted and the internal transcribed spacer (ITS) region (White et al. 1990), ß-tubulin (TUB2) and translation elongation factor 1-alpha (TEF) (Machado et al. 2012) were amplified by PCR, and sequenced. Bayesian inference was used to reconstruct a phylogenetic tree, using MrBayes v. 3.2.1 (Ronquist et al., 2012). The multilocus phylogenetic analysis clearly distinguished the isolate APCG001 as Austropuccinia psidii separating it from all other species. The sequences were deposited in GenBank (accessions nos. ITS: ON003418, TUB2: ON568196, and TEF: ON437601). For pathogenicity tests, four healthy branches (20 leaves each) were sprayed with 2.5 mL of (APCG001) uredospore suspension (105 mL-1) and covered with a plastic bag in the orchard. The air temperature ranged from 16ºC to 25ºC. Sterile distilled water was used as a control. Three replications (pathogen and control) were performed on different trees. After 6 days, symptoms of rust appeared on the plants. Control branches did not show fungal growth. The inoculation test was repeated again, confirming the initial results. This is the first report of infection by A. psidii in C. guazumifolia trees in Brazil, causing rust, necrosis, and early senescence in fruits, leaves, and stems. Myrtaceae rust reduces the C. guazumifolia leaf area, affecting photosynthetic production and reducing fruit quality.

First Report of Myrtle Rust Caused by Austropuccinia psidii on Eugenia myrcianthes in Brazil.

The Eugenia myrcianthes fruit can be consumed in natural or processed form (jellies and juices) (Infante et al., 2016). In 2019, in the UTFPR, Dois Vizinhos city, Paraná State - Brazil, yellow uredinias epiphyllous were observed on the tissue surface (leaves, stems, flowers, and fruit) of twenty-one trees of E. myrcianthes, which resembled structures typical of Myrtaceae rust. All colonized tissues showed necrotic lesions that varied in size and shape, causing death, especially in fruit. In the orchard, the fungus affects 50% to 95% yield. Fruit (10) and leaves (20) with symptoms were collected from 11 trees from different positions in the orchard. Infected tissues were incubated (25°C and 12-hour photoperiod) for 7 days to induce sporulation. The epiphyllous uredinia, united in small groups with hyaline and globose urediniospores were observed and presented equinulate ornaments and germinated pores in the subequatorial and inordinate positions (De Pieri, 2012; Cummins; Hiratsuka, 2003) (mean 14.00 µm × 21.12 µm, n = 30) similar to the morphological characteristics of the Austropuccinia genus described by Young (2019). The identification of 10 samples (fruit and leaf) of the pathogen taken from infected parts of the trees was confirmed. For molecular identification, the internal transcribed spacer (ITS) region was amplified by polymerase chain reaction (White et al. 1990) and sequenced. Phylogenetic analyses using Bayesian inference grouped the strain from Eugenia myrcianthes with the epitype species of Austropuccinia psidii (Beenken, 2017), with a high posterior probability (0.99). The sequences of one representative strain (Emg1) were submitted to GenBank (OM948983). For pathogenicity tests, three healthy branches containing 20 leaves were sprayed with 3.0 mL of urediniospores suspension (105) of Emg1 and covered with a plastic bag in the orchard (25ºC). Sterile distilled water was used as a control. Three replications (pathogen and control) were performed on different trees. After 6 days, symptoms appeared and their morphological features were similar to those previously reported. Control branches did not present fungal growth. The inoculation test was repeated again, confirming symptoms such as uredinia and urediniospores, characteristic of the disease. This is the first report of the incidence of A. psidii infection in E. myrcianthes trees in Brazil, causing rust, necrosis, and senescence in fruit, leaves, flowers, and stems. The rust on E. myrcianthes causes destructive damage to yield, as the pathogen causes fruit to rot and drop prematurely.

First Report of Epicoccum nigrum Causing Leaf Spot of Eugenia involucrata in Brazil.

Eugenia involucrata (DC) is a native fruit species of forest formations in the Atlantic Complex and in the forests and savannas of the Paraná State, Brazil (Donadio, 2002). In February 2021, in the experimental area at the Universidade Tecnológica Federal do Paraná, in the Dois Vizinhos city, Paraná State, Brazil, a foliar disease was observed on twenty-two12 years old E. involucrata trees, with 20 to 80% of the leaves per tree affected. Symptoms were small, irregularly to circular shaped, reddish-brown lesions with yellow halos. As the disease progressed, the lesions increased in size and showed no distinction between mature and young tissues. Twenty symptomatic leaves (from each tree) from 11 trees grown at different locations in the orchard (50% of the total number of trees) were collected. For fungal isolation, the leaf surfaces were disinfected with 0.5% NaOCl solution for 1 min, rinsed in sterile distilled water (SDW) and dried on sterile filter paper. Five fragments (0.3 cm) of diseased leaf tissue were placed on potato dextrose agar medium. After 7 days of incubation at 25°C, orange colonies appeared, with a reddish pigment on the reverse side. Conidial were brown globular to pear solitary, verrucous and multicellular (average of 21.74 µm x 24.45 µm, n = 30). The morphological characteristics of the colony and conidia of the eight isolates matched the description of the fungal genus Epicoccum (Valenzuela-Lopez et al. 2018). Further identification of eight isolates was confirmed by amplifying and sequencing three phylogenic loci (ITS, ß- tubulin and RPB2) using the ITS1/ITS4, Bt2a/Bt2b and 5F2/7cR primer pairs, respectively (White et al., 1991, Glass and Donaldson, 1995, O Donnell et al., 1998). The sequences of one representative isolate (ENcm) were submitted to GenBank (ITS, MZ442338, ß-tubulin, MZ447127 and RPP2, MZ447128) respectively. A phylogenetic tree was constructed by the maximum likelihood method with 1,000 replicas of bootstrapping based on concatenated ITS, ß-tubulin, and RPB2 sequences of the ENcm and strains of 14 species of the genus Epicoccum. Isolate ENcm grouped with Epicoccum nigrum stains CBS 173.73 (Chen et al., 2017). For the pathogenicity tests four young healthy branches containing 20 leaves were spray inoculated, with 1.5 mL of conidia suspension of ENcm (106 conidia/mL) covered with a punched transparent plastic bag and moistened with distilled water in orchard. The air temperature ranged from 14ºC to 25ºC. SDW was used as control. Three replicates (pathogen and control) on different trees were performed. After 7 days the fungus was re-isolated from the symptomatic lesion, showing morphological characteristics similar to those of ENcm. Control branches did not show fungal growth. The inoculation test was repeated once, confirming the symptoms described above. This is the first report of the leaf spot caused by E. nigrum on E. involucrata in Brazil as well as in the world. E. nigrum on E. involucrata leaves could pose potential threat on productivity, whose impact may affect the fruit tree's ability to perpetuate, its survival in natural conditions or in commercial orchards.

Grafting technique and rootstock species for the propagation of Plinia cauliflora / Enxertia e espécie de porta-enxerto na propagação de Plinia cauliflora

ABSTRACT: One of the biggest challenges for expansion of jabuticaba fruit tree cultivation is the high cost of seedlings due to to difficulties with vegetative propagation. Here, we aimed to evaluate graft survival in combinations of Plinia cauliflora and rootstocks of other species from the Myrtaceae family. The study was carried out at the Universidade Tecnológica Federal do Paraná - Campus Dois Vizinhos, Paraná State, Brazil. Eugenia uniflora L., E. involucrata DC, and P. cauliflora were used as rootstocks and were obtained as seeds. The 'whip-and- tongue' and cleft grafting techniques were tested for vegetative propagation. Grafted plants were cultivated in a greenhouse. The experimental design was a randomized complete block, in factorial 3×2 (rootstock species× graft type), with four replicates of 10 grafts by plot. Compatibility between rootstock species and scion was evaluated by diameter growth below and above the grafting point and by vegetative budburst and survival percentages. The use of P. cauliflora grafts with the same species was required for propagation. E. involucrata and E. uniflora rootstocks were not capable of propagation with P. cauliflora . Therefore, it is necessary to test other species from the Myrtaceae family.

RESUMO: Um dos maiores problemas enfrentados para a expansão do cultivo de jabuticabeira é o alto custo das mudas, principalmente, pela dificuldade de multiplicá-las vegetativamente. O objetivo deste trabalho foi analisar a sobrevivência da combinação da enxertia entre jabuticabeira Açú ( Plinia cauliflora ) sobre portas-enxerto de outras espécies da família Myrtaceae . O trabalho foi realizado na UTFPR - Campus Dois Vizinhos (PR). Foi usada a pitangueira ( Eugenia uniflora L.), a cerejeira-do-mato ( Eugenia involucrata DC.) e a própria jabuticabeira Açú ( P. cauliflora ) como porta-enxertos, sendo estes obtidos por meio de sementes. Foram testados dois tipos de enxertia, um a fenda cheia e outro inglês com entalhe. As plantas enxertadas foram mantidas em casa-de-vegetação. O delineamento experimental foi em blocos completamente casualizados, em fatorial 3x2 (espécie de porta-enxerto x tipo de enxertia), com quatro repetições, considerando-se o uso de dez enxertos por parcela. A compatibilidade entre as espécies foi avaliada com base na aferição do crescimento entre os diâmetros do porta-enxerto e enxerto, abaixo e acima do ponto de enxertia, da brotação e a sobrevivência dos enxertos. As enxertias de jabuticabeira Açú com uso de porta-enxerto de mesma espécie foi indicado para sua propagação. Os portas-enxerto de pitangueira e cerejeira da mata não foram indicados, devendo testar outras espécies da família Myrtaceae.

A quitosana como fungistático no crescimento micelial de Rhizoctonia solani Kuhn / Chitosan as fungistatic mycelial growth of Rhizoctonia solani Kuhn

Rhizoctonia solani é um fungo causador de tombamento de plântulas em várias espécies vegetais. A quitosana é um polímero derivado do processo de desacetilação da quitina, a qual é encontrada em grande quantidade na carapaça de crustáceos, insetos e parede celular de fungos. A quitosana tem sido testada para diversos usos, inclusive no controle de fitopatógenos em agricultura, já que apresenta atividade antimicrobiana, para controle de patógenos. O presente trabalho teve como objetivo avaliar o efeito fungistático de diferentes concentrações de quitosana (0; 0,25; 0,5; 1 e 2%) no crescimento micelial do fungo R. solani in vitro. Os resultados obtidos demonstraram efeito significativo de quitosana nas diferentes concentrações utilizadas, na redução do crescimento micelial de R. solani. Observou-se também aumento do efeito fungistático da quitosana conforme o aumento da dose.

Rhizoctonia solani is a fungus that causes damping-off of seedlings in various plant species. Chitosan is a polymer derived from the process of desacetylation of chitin, which is found in large quantities in the exoskeleton of crustaceans, insects and fungal cell wall. Chitosan has been tested for various uses, including the control of plant pathogens in agriculture, since it presents antimicrobial activity to control pathogens. This study aimed to evaluate the fungistatic effect of different chitosan concentrations (0; 0.25; 0.5; 1 and 2%) in mycelial growth in vitro of the fungus R. solani. The results showed a significant effect of different concentrations of chitosan, in reduccing the mycelial growth of R. solani. It was also observed increased fungistatic effect with increasing of the concentration.