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 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 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 Colletotrichum chrysophilum Causing Papaya Anthracnose in Mexico.

Anthracnose, caused by Colletotrichum spp., is the most important fungal disease of papaya (Carica papaya L.) worldwide. In March 2020, mature papaya fruit (cv. Maradol) showing typical symptoms of anthracnose were observed in an orchard located in Pinotepa Nacional, Oaxaca, Mexico. Disease incidence of 100 papaya plants surveyed in the orchard was estimated at about 45%. Initially, small and water-soaked lesions appeared on the fruit surface, which later enlarged to circular sunken lesions with translucent light brown margins. On advanced infections, salmon-pink masses of spores were observed on the lesions. Twenty Colletotrichum-like colonies were consistently isolated on potato dextrose agar (PDA) medium at 25°C in the dark for 6 days and 10 monoconidial isolates were obtained. An isolate was selected as representative for further characterization. The isolate was deposited as CPM-H4 in the Culture Collection of Phytopathogenic Fungi of Plant Pathology Laboratory of the CIIDIR-Oaxaca of the Instituto Politécnico Nacional. On PDA, the colonies were initially light grey then later became dark grey with orange conidial masses after incubation for 7 days. Conidia (n= 50) were hyaline, aseptate, cylindrical with rounded ends, and measured 10.2 to 13.6 × 4.1 to 5.3 µm. Appressoria (n= 20) were mostly simple, solitary and smooth-walled, dark brown, and clavate, measuring 6.8 to 14.8 × 5.5 to 7.7 µm. Based on morphology, the isolate was tentatively identified as belonging to the Colletotrichum gloeosporioides species complex (Weir et al. 2012). For molecular identification, total DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), and partial sequences of actin (ACT), ß-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and chitin synthase (CHS-1) genes were amplified (Weir et al. 2012), and sequenced. The sequences were deposited in GenBank (accessions nos. OM965612 (ITS), OM959540 (ACT), ON065005 (TUB2), ON065003 (CHS-1), ON065004 (GAPDH). A phylogenetic tree based on Bayesian inference and including published ITS, ACT, TUB2, GAPDH, and CHS-1 sequence dataset for Colletotrichum spp. was constructed. The multilocus phylogenetic analysis clearly distinguished the isolate CPM-H4 as Colletotrichum chrysophilum. Pathogenicity of the fungus was verified on 10 healthy papaya fruits (cv. Maradol) without wounds. A drop of a conidial suspension (1 × 105 spores/ml) was placed on three locations on each fruit. Ten control fruit were treated in the same way but with sterilized water. The fruits were kept in a moist plastic chamber at 25°C and 12 h light/dark for 8 days. The pathogenicity test was repeated twice. All inoculated papaya fruits developed sunken necrotic lesions 6 days after inoculation, whereas no symptoms were observed on the control fruits. The fungus was consistently re-isolated only from the diseased fruits and found to be morphologically identical to the isolate used for inoculation, fulfilling Koch´s postulates. Colletotrichum chrysophilum has been previously reported to cause anthracnose on mango (Fuentes-Aragón et al. 2020a), avocado (Fuentes-Aragón et al. 2020b), and banana (Fuentes-Aragón et al. 2021) in Mexico; however, to our knowledge, this is the first report of C. chrysophilum causing papaya anthracnose in Mexico. Therefore, it is necessary to explore the diversity of Colletotrichum species associated with papaya in Mexico through subsequent phylogenetic studies as well as to monitor the possible movement and distribution of this pathogen into other Mexican regions.

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.

Stepwise stress testing of different CAD-CAM lithium disilicate veneer application methods applied to lithium disilicate substructures.

STATEMENT OF PROBLEM: Whether a computer-aided design and computer-aided manufacture (CAD-CAM) fabricated high-translucency lithium disilicate veneer on a lithium disilicate substructure would increase the strength of the restoration compared with a traditional feldspathic porcelain veneer is unclear. PURPOSE: The purpose of this in vitro study was to evaluate the effect of different lithium disilicate veneer application methods on a lithium disilicate substructure on their biaxial flexural stress (BFS). MATERIAL AND METHODS: Lithium disilicate disks were fabricated so that when combined with the veneering disks, they had a dimension of 12×1.2 mm. Experimental groups were as follows (n=15): resin-bonded lithium disilicate veneer, lithium disilicate veneer adhesively cemented to lithium disilicate; sintered lithium disilicate veneer, lithium disilicate veneer sintered to lithium disilicate; sintered feldspathic veneer, feldspathic porcelain applied to lithium disilicate; and monolithic lithium disilicate, the control group. Weibull distribution survival analysis was used to compare the differences in the resistance to fracture after fatigue. The total number of cycles was analyzed by using 1-way ANOVA (α=.05). A finite element analysis (FEA) was also performed. The maximum principal stress (MPS) was used as the failure criterion. RESULTS: The sintered feldspathic veneer group had significantly lower fatigue resistance than sintered lithium disilicate veneer or resin-bonded lithium disilicate veneer (P<.05). The resin-bonded lithium disilicate veneer group showed significantly more fractured fragments than the other groups. No statistical difference was observed in the number of cycles. The lithium disilicate veneered groups presented similar resistance to fatigue as the monolithic specimens of the same overall dimensions. Higher peaks of MPS were observed for groups monolithic lithium disilicate, sintered lithium disilicate veneer, and sintered feldspathic veneer than for resin-bonded lithium disilicate veneer. CONCLUSIONS: Veneering a lithium disilicate substructure with a lithium disilicate veneer, bonded or sintered, increased resistance to fatigue compared with a feldspathic porcelain veneer. The lithium disilicate veneer groups had similar fatigue resistance to that of the monolithic group.

First Report of Colletotrichum siamense Causing Anthracnose of Guava (Psidium guajava) in Mexico.

Guava (Psidium guajava L.) is a small tree belonging to the Myrtaceae family and it is distributed worldwide in the tropical and subtropical areas. During the summer of 2019, symptoms of fruit anthracnose were observed on approx. 90% of 250 guava trees located in backyards in Juan Jose Rios, Sinaloa, Mexico. Lesions on guava fruit were irregular, necrotic, and sunken. On advanced infections, acervuli containing salmon-pink masses of spores were observed on the lesions. Twenty fruits were collected from 10 trees (2 fruits per tree). Colletotrichum-like colonies were consistently isolated on PDA medium and 20 monoconidial isolates were obtained. Four isolates were selected as representatives for morphological characterization, multilocus phylogenetic analysis, and pathogenicity tests. The isolates were deposited in the Culture Collection of Phytopathogenic Fungi of the Faculty of Agriculture of El Fuerte Valley at the Sinaloa Autonomous University (Accession nos. FAVF205-FAVF208). Colonies on PDA medium were flat with an entire margin, with abundant felty and white aerial mycelium, with pink conidial masses. Conidia (n= 100) were cylindrical, hyaline, aseptate, with ends rounded, and measuring 14.8 to 18.1 × 4.4 to 5.3 µm. Based on morphological features, the isolates were tentatively allocated in the C. gloeosporioides species complex (Weir et al. 2012). For molecular identification, genomic DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), as well as partial sequences of actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ß-tubulin (TUB2), chitin synthase (CHS-1) and glutamine synthetase (GS) genes were amplified by PCR (Weir et al. 2012), and sequenced. A phylogenetic tree based on Bayesian inference and including published ITS, GAPDH, TUB2, ACT, CHS-1, and GS data for Colletotrichum species was constructed. The multilocus phylogenetic analysis clearly distinguished the four isolates FAVF205-FAVF208 as C. siamense separating it from all other species within the C. gloeosporioides species complex. The sequences were deposited in GenBank (accessions nos. ITS: MW598512-MW598515; GAPDH: MW595216-MW595219; TUB2: MW618012-MW618015; ACT: MW595208-MW595211; CHS-1: MW595212-MW595215; and GS: MW618008-MW618011). Pathogenicity of the four isolates was verified on 40 healthy guava fruits. Twenty fruits were wounded with a sterile toothpick (2 mm in depth) and a mycelial plug (6 mm of diameter) was placed on each wound. Ten fruits inoculated with a PDA plug without mycelial growth served as controls. The fruit was kept in a moist plastic chamber at 25°C for 7 days. Pathogenicity of each isolate was tested with both non-wound and wound inoculation methods. The experiments were repeated twice with similar results. All inoculated fruits developed sunken necrotic lesions 4 days after inoculation, whereas no symptoms were observed on the control fruits. The fungi were consistently re-isolated only from the diseased fruits, fulfilling Koch´s postulates. Colletotrichum siamense has been previously reported on guava fruit in India (Sharma et al. 2015). However, to our best knowledge, this is the first report of C. siamense causing fruit anthracnose on guava in Mexico. Therefore, it is necessary to explore the diversity of Colletotrichum species on guava in detail through subsequent phylogenetic studies as well as to monitor the distribution of this pathogen into other Mexican regions.

Influence of S. cerevisiae and P. kluyveri as starters on chocolate flavour.

BACKGROUND: Fermented cocoa beans (Theobroma cacao L.) are a pivotal raw material for chocolate production. A cocktail yeast applied in the cocoa fermentation process can promote the formation of pleasant metabolites. Saccharomyces, Pichia and Hanseniaspora have been widely used in fermentation to improve the final product organoleptic profile, highlighting that fermentation is a critical point for chocolate flavour precursor production. This study aims to evaluate the impact of Pichia kluyveri and Saccharomyces cerevisiae strains as starter cultures on the fermentation for two cocoa hybrids, FA13 and CEPEC2002. RESULTS: During fermentation processes, volatile organic compounds (VOCs) and protein profiles were assessed. Chocolates produced were also assessed regarding the presence of VOCs. Eighty VOCs were identified using gas chromatography coupled to mass spectrometry analysis. Mass spectrometry provided the protein profile evolution during fermentation and showed that the profiles changed with inoculation type (spontaneous versus inoculated fermentation). Chocolate obtained from FA13 inoculated with S. cerevisiae strain contained a greater amount of organics acids, being categorised as sourer than chocolate produced by spontaneous fermentation of FA13. CEPEC2002 inoculated with S. cerevisiae strain in co-culture with P. kluyveri strain generated less sour and sweeter chocolate than spontaneous fermentation only. CONCLUSIONS: Chocolates from inoculated assays with starter cultures were more accepted by evaluators, highlighting that P. kluyveri and S. cerevisiae influence the composition of VOCs. Besides, protein profiles also changed throughout fermentation. Further investigation should be conducted to clarify protein degradation dynamics during inoculated fermentations to define which of the microbial cultures positively affect the chocolate sensory characteristics. © 2021 Society of Chemical Industry.

Gongronella eborensis sp. nov., from vineyard soil of Alentejo (Portugal).

This study describes a novel fungal species belonging to the genus Gongronella. During a previous work focusing on metalaxyl degradation by Mucorales strains, two isolates from vineyard soil samples collected in the Alentejo region, south Portugal, were identified as a putative novel species based on combined molecular and MALDI-TOF MS data. This new species is described here using a polyphasic approach that combines morphology, internal transcribed spacer of ribosomal DNA (ITS) and 28S ribosomal DNA (LSU) sequence data analysis and proteomic profiling by MALDI-TOF MS. Phenotypic and molecular data enabled this novel species to be clearly distinguished from other Gongronella species with results of combined ITS+LSU analysis showing that the Gongronella species is related to Gongronella butleri and Gongronella brasiliensis. Therefore, from the results of morphological and molecular analyses, isolates MUM 10.262 and MUM 10.263 seem to represent a new Gongronella species and the name Gongronella eborensis sp. nov. is proposed, with the ex-type strain MUM 10.262 (=CCMI 1100=CBS 128763).

Effect of quorum sensing and quenching molecules on inter-kingdom biofilm formation by Penicillium expansum and bacteria.

The ecology of a biofilm is a complex function of different factors, including the presence of microbial metabolites excreted by the inhabitants of the biofilm. This study aimed to assess the effect of patulin, and N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) on inter-kingdom biofilm formation between a filamentous fungus and bacteria isolated from drinking water. The filamentous fungus Penicillium expansum and the bacteria Acinetobacter calcoaceticus and Methylobacterium oryzae were used as model species. M. oryzae biofilm formation and development was more susceptible to the presence of the quenching molecules than A. calcoaceticus biofilms. Patulin reduced M. oryzae biofilm growth while 3-oxo-C12-HSL caused an increase after 48 h. The presence of P. expansum had a detrimental effect on M. oryzae cell numbers, while an advantageous effect was observed with A. calcoaceticus. The overall results reveal that quorum sensing and quenching molecules have a significant effect on inter-kingdom biofilm formation, especially on bacterial numbers.

First Report of Colletotrichum siamense and C. gloeosporioides Causing Anthracnose of Citrus spp. in Mexico.

Citrus anthracnose, caused by Colletotrichum spp., is a major disease in many citrus-growing regions of the world. During the spring of 2019, symptoms of petal necrosis and necrotic lesions on fruits were detected on Mexican lime (Citrus aurantifolia), sweet orange (Citrus sinensis), and grapefruit (Citrus paradisi) trees in three commercial orchards distributed in northern Sinaloa (El Fuerte and Ahome municipalities), Mexico. Colletotrichum-like colonies were consistently isolated on potato dextrose agar (PDA) medium from symptomatic petals and fruits, and 30 monoconidial isolates (10 per orchard) were obtained. Five isolates were selected as representative for morphological characterization, multilocus phylogenetic analysis, and pathogenicity tests. The isolates were designated as FAVF355-FAVF359 and were deposited in the Culture Collection of Phytopathogenic Fungi of the Faculty of Agronomy of El Fuerte Valley at the Autonomous University of Sinaloa (Mexico). Colonies grown on PDA at 25ºC were cottony, dense, with grayish white aerial mycelium and with pink conidial masses. Conidia (n= 100) were cylindrical, hyaline, aseptate, 13.7 to 18.8 × 4.3 to 5.8 µm, with both ends rounded. Based on morphological features, the five isolates were tentatively identified in the Colletotrichum gloeosporioides species complex (Weir et al. 2012). For molecular identification, total DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), and partial sequences of actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ß-tubulin (TUB2) genes were amplified by PCR (Weir et al. 2012), and sequenced. A phylogenetic tree based on Bayesian inference for species belonging to the C. gloeosporioides species complex was constructed. The multilocus phylogenetic analysis distinguished the isolates FAVF355-FAVF357 as C. gloeosporioides sensu stricto and the isolates FAVF358-FAVF359 as C. siamense. The sequences were deposited in GenBank (accession numbers ITS: MT850050-MT850054; ACT: MT834528-MT834532; GAPDH: MT855979-MT855982; TUB2: MT834533-MT834536). Pathogenicity of the five isolates was verified on healthy fruits of their original host species. Five fruits per isolate were inoculated using the colonized agar plug method. Fruits were wounded with a sterile toothpick and mycelial plugs (5 mm in diameter) removed from the margin of a 6-days-old culture were placed onto three wound sites in each fruit. Non-colonized agar plugs were placed on the wounds of 10 fruits used as the control. The fruits were kept in a moist chamber at 25°C for 8 days. The experiment was repeated twice. All inoculated fruits developed circular and necrotic lesions 6 days after inoculation, whereas the control fruits remained symptomless. The fungi were consistently re-isolated from the diseased fruits and were morphologically identical to that originally inoculated, fulfilling Koch´s postulates. To date, only C. gloeosporioides sensu lato and C. acutatum sensu lato has been associated with sweet orange and Mexican lime in Mexico (Farr and Rossman 2020), whereas C. gloeosporioides sensu stricto has been recently recorded in a different area (Iguala, Guerrero) of Mexico (Cruz-Lagunas et al. 2020). To our knowledge, this is the first report of C. gloeosporioides sensu stricto causing anthracnose on sweet orange, and of C. siamense on Mexican lime in Mexico, as well as C. gloeosporioides s. s. causing disease on grapefruit in Sinaloa, Mexico.

First Report of Colletotrichum jiangxiense Causing Avocado Anthracnose in Mexico.

Mexico is the largest avocado (Persea americana) producer and exporter in the world. In January of 2019, typical symptoms of fruit anthracnose were observed on approximately 90% of avocado trees in backyards localized in Leonardo Bravo municipality in Guerrero, Mexico. Lesions on avocado fruits were circular, necrotic, and sunken, whereas the mesocarp showed a soft rot with dark brown discoloration. To perform fungal isolation, small pieces from adjacent tissue to lesions of five symptomatic fruits were surface disinfested by immersion in a 1% sodium hypochlorite solution for 2 min, rinsed in sterile distilled water, and placed in Petri dish containing potato dextrose agar (PDA). Plates were incubated at 25 ºC for 5 days in darkness. Colletotrichum-like colonies were consistently isolated and seven monoconidial isolates were obtained. An isolate was selected as a representative for morphological characterization, molecular analysis, and pathogenicity tests. The isolate was deposited in the Culture Collection of Phytopathogenic Fungi at the Colegio Superior Agropecuario del Estado de Guerrero (Accession No. CSAEG-CJ19). After 8 days on PDA, the colonies were gray on the upper surface, and with orange conidial masses. Conidia (n= 100) were cylindrical, hyaline, aseptate, with rounded ends, 14.4 to 18.5 × 4.5 to 6.2 µm. Based on morphological features, the isolate was tentatively identified in the C. gloeosporioides species complex (Weir et al. 2012). For molecular identification, genomic DNA was extracted and the internal transcribed spacer (ITS) region of rDNA, and partial sequences of actin (ACT), ß-tubulin (TUB2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified by PCR, and sequenced with primers ITS5/ITS4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn 1999), Bt2A/Bt2B (Glass and Donaldson 1995), and GDF/GDR (Templeton et al. 1992), respectively. BLAST analysis of the obtained sequences of the ITS, ACT, TUB2, and GAPDH genes revealed 100%, 99.63%, 99.77% and 100% identity with those of isolate LF687 of C. jiangxiense in GenBank (Accession numbers KJ955201, KJ954471, KJ955348, and KJ954902). A phylogenetic tree based on Bayesian inference and including published ITS, ACT, TUB2, and GAPDH data for Colletotrichum species was constructed. The multilocus phylogenetic analysis clearly distinguished the isolate CSAEG-CJ19 as C. jiangxiense separating it from all other species within the C. gloeosporioides species complex. The sequences were deposited in GenBank (accession numbers ITS:MT011397; ACT:MN968784, TUB2:MN968786, and GAPDH:MN968785). To conduct Koch's postulates, 20 healthy avocado fruits (cv. Hass) were wounded with a sterile toothpick (2 mm in depth) and a drop of 15 µl of conidial suspension (1 × 105 spores/mL) was placed on each wound. Ten control fruit were wounded and treated with sterilized water. All the fruits were kept in a moist plastic chamber at 25°C for 8 days. All inoculated fruits developed circular and necrotic lesions (12 to 18 mm in diameter), 5 days after inoculation, whereas control fruits remained healthy. The fungus was consistently re-isolated from the inoculated fruits. Previously, C. jiangxiense has been reported as a pathogen on Camellia sinensis and Citrus sinensis in China (Farr and Rossman 2020). To our knowledge, this is the first report of C. jiangxiense causing anthracnose on avocado worldwide. This study shown another species in the C. gloeosporioides complex associated with avocado diseases in Mexico. Therefore, it is necessary to explore the diversity of Colletotrichum species in detail through subsequent phylogenetic studies as well as to monitor the distribution of this pathogen into other Mexican regions.

In vitro assessment of inter-kingdom biofilm formation by bacteria and filamentous fungi isolated from a drinking water distribution system.

The main focus so far in the study of biofilm formation in drinking water has been bacteria. Studies on biofilm formation involving filamentous fungi are, therefore, scarce. This study aimed to assess and characterize the ability of these microorganisms to interact with bacteria whilst forming inter-kingdom biofilms. Biofilms were analysed in terms of total biomass, metabolic activity, bacterial colony forming units and morphology by epifluorescence microscopy. The quantitative methods revealed that biofilm mass increased over time for both single and inter-kingdom biofilms, while specific metabolic activity decreased, in general, along the time points evaluated. Microscopic data visually confirmed the biofilm mass increase over time. This study shows that fungal stage development is important in the first 24 h of biofilm formation. Inter-kingdom biofilm formation is microorganism dependent and inter-kingdom biofilms may provide an advantage to the opportunistic bacterium Acinetobacter calcoaceticus to replicate and proliferate when compared with Methylobacterium oryzae.

World data centre for microorganisms: an information infrastructure to explore and utilize preserved microbial strains worldwide.

The World Data Centre for Microorganisms (WDCM) was established 50 years ago as the data center of the World Federation for Culture Collections (WFCC)-Microbial Resource Center (MIRCEN). WDCM aims to provide integrated information services using big data technology for microbial resource centers and microbiologists all over the world. Here, we provide an overview of WDCM including all of its integrated services. Culture Collections Information Worldwide (CCINFO) provides metadata information on 708 culture collections from 72 countries and regions. Global Catalogue of Microorganism (GCM) gathers strain catalogue information and provides a data retrieval, analysis, and visualization system of microbial resources. Currently, GCM includes >368 000 strains from 103 culture collections in 43 countries and regions. Analyzer of Bioresource Citation (ABC) is a data mining tool extracting strain related publications, patents, nucleotide sequences and genome information from public data sources to form a knowledge base. Reference Strain Catalogue (RSC) maintains a database of strains listed in International Standards Organization (ISO) and other international or regional standards. RSC allocates a unique identifier to strains recommended for use in diagnosis and quality control, and hence serves as a valuable cross-platform reference. WDCM provides free access to all these services at www.wdcm.org.

Penicillium tunisiense sp. nov., a novel species of Penicillium section Ramosa discovered from Tunisian orchard apples.

Two similar Penicillium isolates could not be identified as previously described species in a survey of orchard apples from Tunisia for patulin-producing fungi. These isolates are described as novel species using multilocus DNA sequence analysis of partial ß-tubulin, calmodulin and nuclear ribosomal internal transcribed spacer regions; and morphological, physiological and biochemical characteristics. The isolates were considered negative for patulin production since the IDH gene fragment was not detected and the compound detected at the same retention time of patulin (14.9 min) showed a different UV spectrum using U-HPLC/UV-DAD. In terms of phylogeny, the two isolates clustered with Penicillium section Ramosa and are closely related to Penicillium chroogomphum, Penicillium lenticrescens and Penicillium soppii. Furthermore, their macro- and micromorphological traits differed from these species. Hence, the isolates represent a novel species in Penicillium section Ramosa and the name Penicillium tunisiense sp. nov. is proposed, with the type strain MUM 17.62T (=ITEM 17445T).

Collagen content in the bladder of men with LUTS undergoing open prostatectomy: A pilot study.

AIMS: To evaluate the collagen content in the bladder wall of men undergoing open prostate surgery. METHODS: From July 2014 to August 2016, men aged ≥ 50 years, presenting LUTS and undergoing open prostate surgery due to benign prostatic enlargement (BPE) or prostate cancer were prospectively enrolled. Preoperative assessment included validated questionnaires (IPSS and OAB-V8), lower urinary tract ultrasound, and urodynamics. Bladder biopsies were obtained during open prostatectomy for determination of collagen content (sirius red-picric acid stain; polarized light analysis). Collagen to smooth muscle ratio (C/M) in the detrusor was measured and its relationship with preoperative parameters was investigated. The level of significance was P < 0.05. RESULTS: Thirty-eight consecutive patients were included in this pilot study. Mean age was 66.36 ± 6.44 years and mean IPSS was 11.05 ± 8.72 points. Men diagnosed with diabetes mellitus (DM2) were found to have higher collagen content in the bladder wall when compared to non-diabetic patients (17.71 ± 6.82% vs 12.46 ± 5.2%, respectively; P = 0.024). Reduced bladder compliance was also marker for higher collagen content (P = 0.042). Bladder outlet obstruction (BOO) was not a predictor of increased collagen deposition in the bladder wall (P = 0.75). Patients with PVR ≥ 200 mL showed a higher collagen to smooth muscle ratio in the bladder wall (P = 0.036). CONCLUSIONS: DM2 and urodynamic parameters, such as increased PVR and reduced bladder compliance, were associated with higher collagen content in the bladder wall of men with LUTS.

Polyphasic identification of Penicillia and Aspergilli isolated from Italian grana cheese.

Penicillium and Aspergillus genera, both including mycotoxin producing species, were reported as associated to cheese and cheese working environment, but never studied in an extensive way in Italian grana cheese (Grana Padano and Parmigiano Reggiano). The aim of this work was to address the identification of Aspergilli and Penicillia associated to grana cheese in order to lay down the basis for risk assessment and safe processing for a high quality production. One hundred and four strains belonging to Aspergillus and Penicillium genera were obtained from cheese crust and from ripening room air (with the latter largely dominant), and identified following a polyphasic approach, strongly required for the identification at the species level. Morphological observation was used along with molecular techniques, RAPD-PCR fingerprinting and calmodulin gene sequencing (CaM), the former aimed to limit as much as possible the latter sequencing effort. Seventy four percent of the strains were assigned to Penicillium subgenus Penicillium, section Fasciculata. Main mycotoxin producing species identified were A. flavus, P. crustosum and P. verrucosum, while the dominant species in both air and cheese crust was P. solitum, which has never been so far reported as mycotoxigenic. Results obtained in this study confirmed that mycotoxin contamination is a possible issue to face during grana cheese making.

Oxidative stress in the bladder of men with LUTS undergoing open prostatectomy: a pilot study.

PURPOSE: This study aims to evaluate the link between preoperative parameters and oxidative stress (OS) markers in the bladder wall of men undergoing open prostatectomy. MATERIALS AND METHODS: From July 2014 to August 2016, men aged ≥ 50 years and presenting with LUTS were prospectively enrolled. Preoperative assessment included validated questionnaires (IPSS and OAB - V8), lower urinary tract ultrasound and urodynamics. Bladder biopsies were taken during open prostatectomy for determination of OS markers. Increased OS was defined by increased concentration of malondialdehyde (MDA) and / or decreased concentration of antioxidant enzymes (superoxide dismutase and / or catalase). P<0.05 was regarded as statistically significant. RESULTS: Thirty - eight consecutive patients were included. Mean age was 66.36 ± 6.44 years, mean prostate volume was 77.7 ± 20.63 cm3, and mean IPSS was 11.05 ± 8.72 points. MDA concentration was increased in men with severe bladder outlet obstruction (BOO grade V - VI according to the Schaefer's nomogram) in comparison with BOO grade III - IV (p = 0.022). Patients with severe LUTS also had higher MDA concentration when compared to those with mild LUTS (p = 0.031). There was a statistically significant association between increased post - void residual urine (cut off ≥ 50 mL) and not only higher levels of MDA, but also reduced activity of SOD and catalase (p < 0.05). CONCLUSIONS: This pilot study showed that severity of LUTS and BOO were associated with increased MDA concentration in the bladder wall of men undergoing open prostatectomy. Further studies are still needed to assess the role of non - invasive biomarkers of OS in predicting bladder dysfunction in men with LUTS.

Metalaxyl Degradation by Mucorales Strains Gongronella sp. and Rhizopus oryzae.

In this study, the degradation of metalaxyl was investigated in the presence of two Mucorales strains, previously isolated from soil subjected to repeated treatments with this fungicide and selected after enrichment technique. Fungal strains were characterised by a polyphasic approach using phylogenetic analysis of the Internal Transcribed Spacer (ITS) gene region, phenotypic characterisation by Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) spectral analysis, and growth kinetics experiments. The strains were identified as Gongronella sp. and Rhizopus oryzae. The fungal growth kinetics in liquid cultures containing metalaxyl fits with Haldane model. Under laboratory conditions, the ability of Gongronella sp. and R. oryzae cultures to degrade metalaxyl was evaluated in liquid cultures and soil experiments. Both species were able to: (a) use metalaxyl as the main carbon and energy source; and (b) degrade metalaxyl in polluted soils, with rates around 1.0 mg kg-¹ d-¹. This suggests these strains could degrade metalaxyl in soils contaminated with this fungicide.

Impact of a Microbial Cocktail Used as a Starter Culture on Cocoa Fermentation and Chocolate Flavor.

Chocolate production suffered a vast impact with the emergence of the "witches' broom" disease in cocoa plants. To recover cocoa production, many disease-resistant hybrid plants have been developed. However, some different cocoa hybrids produce cocoa beans that generate chocolate with variable quality. Fermentation of cocoa beans is a microbiological process that can be applied for the production of chocolate flavor precursors, leading to overcoming the problem of variable chocolate quality. The aim of this work was to use a cocktail of microorganisms as a starter culture on the fermentation of the ripe cocoa pods from PH15 cocoa hybrid, and evaluate its influence on the microbial communities present on the fermentative process on the compounds involved during the fermentation, and to perform the chocolate sensorial characterization. According to the results obtained, different volatile compounds were identified in fermented beans and in the chocolate produced. Bitterness was the dominant taste found in non-inoculated chocolate, while chocolate made with inoculated beans showed bitter, sweet, and cocoa tastes. 2,3-Butanediol and 2,3-dimethylpyrazine were considered as volatile compounds making the difference on the flavor of both chocolates. Saccharomyces cerevisiae UFLA CCMA 0200, Lactobacillus plantarum CCMA 0238, and Acetobacter pasteurianus CCMA 0241 are proposed as starter cultures for cocoa fermentation.