First report of Phyllosticta citricarpa and description of two new species, P. paracapitalensis and P. paracitricarpa, from citrus in Europe.

The genus Phyllosticta occurs worldwide, and contains numerous plant pathogenic, endophytic and saprobic species. Phyllosticta citricarpa is the causal agent of Citrus Black Spot disease (CBS), affecting fruits and leaves of several citrus hosts (Rutaceae), and can also be isolated from asymptomatic citrus tissues. Citrus Black Spot occurs in citrus-growing regions with warm summer rainfall climates, but is absent in countries of the European Union (EU). Phyllosticta capitalensis is morphologically similar to P. citricarpa, but is a non-pathogenic endophyte, commonly isolated from citrus leaves and fruits and a wide range of other hosts, and is known to occur in Europe. To determine which Phyllosticta spp. occur within citrus growing regions of EU countries, several surveys were conducted (2015-2017) in the major citrus production areas of Greece, Italy, Malta, Portugal and Spain to collect both living plant material and leaf litter in commercial nurseries, orchards, gardens, backyards and plant collections. A total of 64 Phyllosticta isolates were obtained from citrus in Europe, of which 52 were included in a multi-locus (ITS, actA, tef1, gapdh, LSU and rpb2 genes) DNA dataset. Two isolates from Florida (USA), three isolates from China, and several reference strains from Australia, South Africa and South America were included in the overall 99 isolate dataset. Based on the data obtained, two known species were identified, namely P. capitalensis (from asymptomatic living leaves of Citrus spp.) in Greece, Italy, Malta, Portugal and Spain, and P. citricarpa (from leaf litter of C. sinensis and C. limon) in Italy, Malta and Portugal. Moreover, two new species were described, namely P. paracapitalensis (from asymptomatic living leaves of Citrus spp.) in Italy and Spain, and P. paracitricarpa (from leaf litter of C. limon) in Greece. On a genotypic level, isolates of P. citricarpa populations from Italy and Malta (MAT1-2-1) represented a single clone, and those from Portugal (MAT1-1-1) another. Isolates of P. citricarpa and P. paracitricarpa were able to induce atypical lesions (necrosis) in artificially inoculated mature sweet orange fruit, while P. capitalensis and P. paracapitalensis induced no lesions. The Phyllosticta species recovered were not found to be widespread, and were not associated with disease symptoms, indicating that the fungi persisted over time, but did not cause disease.

A Global Perspective on the Population Structure and Reproductive System of Phyllosticta citricarpa.

The citrus pathogen Phyllosticta citricarpa was first described 117 years ago in Australia; subsequently, from the summer rainfall citrus-growing regions in China, Africa, and South America; and, recently, the United States. Limited information is available on the pathogen's population structure, mode of reproduction, and introduction pathways, which were investigated by genotyping 383 isolates representing 12 populations from South Africa, the United States, Australia, China, and Brazil. Populations were genotyped using seven published and eight newly developed polymorphic simple-sequence repeat markers. The Chinese and Australian populations had the highest genetic diversities, whereas populations from Brazil, the United States, and South Africa exhibited characteristics of founder populations. The U.S. population was clonal. Based on principal coordinate and minimum spanning network analyses, the Chinese populations were distinct from the other populations. Population differentiation and clustering analyses revealed high connectivity and possibly linked introduction pathways between South Africa, Australia, and Brazil. With the exception of the clonal U.S. populations that only contained one mating type, all the other populations contained both mating types in a ratio that did not deviate significantly from 1:1. Although most populations exhibited sexual reproduction, linkage disequilibrium analyses indicated that asexual reproduction is important in the pathogen's life cycle.

Canopy structure, ingestive behavior and displacement patterns of beef heifers grazing warm-season pastures / Estrutura do dossel, comportamento ingestivo e padrões de deslocamento de novilhas de corte em pastagens de estação quente

Analyses in a data set of six experiments (n=436) was performed with the aim of characterizing canopy structure and forage intake patterns of beef heifers grazing on Pearl millet (Pennisetum americanum), Alexandergrass (Urochloa plantaginea) and Coastcross (Cynodon dactylon). Forage and leaf lamina mass were similar among species, 3001.4 and 668.1kg of DM/ha, respectively, while leaf:stem ratio of canopy vertical strata was different. Intake rate (12g DM/minute), bite mass (0.343g DM/bite) and bite rate (36.6bites per minute) were similar in Pearl millet and Alexandergrass. In Pearl millet, neutral detergent fiber content (56.1%) in forage as grazed, grazing time (518.9 minutes/day) and displacement rate (8.8 steps/minute) were lower. Increased grazing time (639 minutes/day) represented the main compensatory mechanism for smaller bite masses (0.234g DM/bite) and reduced intake rates (8.8 grams of DM/minutes) in Coastcross. Bite rate variations happened as a response to constraints imposed by canopy structure. Surface utilization on Alexandergrass and Coastcross was increased by greater number of feeding stations visited and displacement rate. Ingestive behavior components of beef heifers are affected by differences in the canopy structure of Pearl millet, Alexandergrass and Coastcross.

Foi realizada a análise conjunta dos resultados de seis experimentos (n=436) com o objetivo de caracterizar a estrutura do dossel e os padrões de ingestão de forragem por novilhas de corte em pastagem de milheto (Pennisetum americanum), papuã (Urochloa plantaginea) e coastcross (Cynodon dactylon). As massas de forragem e de lâminas foliares foram similares entre as três espécies forrageiras, 3001,4 e 668,1kg/ha de MS, respectivamente, enquanto a relação lâmina:colmo do perfil vertical do dossel diferiu. A taxa de ingestão (12g MS/minuto), a massa de bocados (0,343g MS/bocado) e a taxa de bocados (36,6 bocados/minuto) foram similares no milheto e no papuã. O conteúdo de fibra em detergente neutro (56,1%) na forragem da simulação do pastejo, o tempo de pastejo (518,9 minutos/dia) e a taxa de deslocamento (8,8 passos/minuto) foram menores no milheto. O incremento no tempo de pastejo (639 minutos/dia) representou o mecanismo compensatório para a menor massa de bocados (0,234g MS/bocado) e para a reduzida taxa de ingestão (8,8 gramas MS/minuto) no coastcross. Variações na taxa de bocado ocorrem como resposta às limitações impostas pela estrutura do dossel. A exploração da área da pastagem de papuã e de coastcross foi aumentada pelo maior número de estações alimentares visitadas e pela maior taxa de deslocamento. Os componentes do comportamento ingestivo de novilhas de corte são afetados por diferentes estruturas no dossel de milheto, papuã e coastcross.

16S-gyrB-rpoB multilocus sequence analysis for species identification in the genus Microbispora.

The genus Microbispora has been considered difficult to define taxonomically. While 16S rRNA gene analysis is required to determine phylogenetic relationships among species in this genus, most 16S rRNA gene-based phylogenetic tree topologies are not reliable. The genus Microbispora currently contains eight species along with six reclassified species (Microbispora chromogenes, Microbispora diastatica, Microbispora parva, Microbispora indica, Microbispora karnatakensis, Microbispora rosea) and Microbispora rosea subsp. aerata, a taxon composed of three further reclassified species (Microbispora aerata, Microbispora thermodiastatica, and Microbispora thermorosea). 16S rRNA, 23S rRNA, gyrB, and rpoB gene sequences were obtained for the type strains of Microbispora species, and eleven endophytic isolates from a Brazilian medicinal plant, Vochysia divergens. Using the concatenated sequence, most Microbispora type strains could be distinguished with high probability support. Based on these analyses, we propose that five of the species reclassified within the subspecies of M. rosea (M. chromogenes, M. karnatakensis, M. parva, M. aerata and M. thermorosea) are distinct from M. rosea and so should be retained as distinct species. The concatenated 16S-gyrB-rpoB gene phylogenic tree had significant probability support and topology. We propose the use of concatenated 16S-gyrB-rpoB gene sequences to determine phylogenetic relationships within the genus Microbispora. We also suggest that strains sharing >98.1 % 16S-gyrB-rpoB gene sequences similarity be defined as a single species, based on results from this analysis. Seven of the strains isolated from V. divergens were not related to any previously described Microbispora species.

Padrões de deslocamento de bezerras de corte que receberam suplementos isolipídicos em pastagem de azevém / Displacement patterns of beef heifers receiving iso lipidic supplements on ryegrass pasture

Foram avaliados o uso de estações alimentares, o deslocamento e a taxa de bocado de bezerras de corte que foram mantidas exclusivamente em pastagem de azevém (Lolium multiflorum Lam.) ou receberam suplementos (grão de milho ou gordura). O método de pastejo foi contínuo, com número variável de animais. O delineamento experimental foi inteiramente ao acaso, com medidas repetidas no tempo. O número de bocados realizados por estação alimentar é similar quando as bezerras recebem suplemento ou não. As equações de predição mostram que a massa de lâminas foliares exerce maior influência no tempo por estação alimentar quando as bezerras permanecem exclusivamente em pastejo de azevém ou recebem grão de milho como suplemento, enquanto para bezerras que recebem gordura, essa variável é influenciada pela oferta de forragem. O deslocamento (passos entre estações e passos por minuto) de bezerras exclusivamente em pastejo e que recebem grão de milho é influenciado pela estrutura e qualidade do pasto. A taxa de bocado dos animais suplementados com grão de milho e gordura é influenciada pela proporção de lâminas foliares no dossel. Equações de regressão múltipla, considerando-se os atributos do pasto e da pastagem, podem ser utilizadas como modelos preditores do uso de estações alimentares, deslocamento e taxa de bocado de bezerras de corte...

The use of feeding stations, displacement patterns and bite rate of beef heifers kept exclusively on Italian ryegrass (Lolium multiflorum Lam) or receiving supplements (corn grain or fat) were evaluated. The grazing method was continuous with a variable number of animals. The experimental design was a completely randomized design with repeated time measures. The number of bites per feeding station is similar when heifers receive supplement or not. The prediction equations show that the mass of leaf lamina has greater influence on the time per feeding station when heifers remain exclusively grazing ryegrass or receive corn grain as a supplement, while for calves that receive fat this variable is influenced by forage allowance. The displacement (steps between stations and steps per minute) of heifers exclusively on ryegrass or receiving corn grain is influenced by the structure and quality of grass. The bite rate of heifers supplemented with corn grain and fat is influenced by the proportion of leaf lamina in the sward. Multiple regression equations considering the attributes of grass and pasture can be used as models to predict the use of feeding stations, displacement and bite rate of beef heifers...

Produtividade de sistemas forrageiros consorciados com leguminosas / Productivity of grazing systems mixed with forage legumes

O objetivo desta pesquisa foi avaliar três sistemas forrageiros (SF) com capim elefante (CE) + azevém (AZ) + espécies de crescimento espontâneo (ECE); CE + AZ + ECE + amendoim forrageiro (AM); e CE + AZ + ECE + trevo vermelho (TV), usando-se a mesma área, sob pastejo rotacionado, no decorrer do ano agrícola. O CE foi estabelecido em linhas afastadas a cada 4m. No período hibernal, fez-se o estabelecimento do AZ entre as linhas do CE; o TV foi semeado e o AM foi preservado, considerando-se os respectivos SF. O delineamento experimental foi o inteiramente ao acaso, com três tratamentos (SF), duas repetições (piquetes) e avaliações independentes (ciclos de pastejos). Para avaliação, foram utilizadas vacas da raça Holandesa em lactação, que receberam suplementação alimentar com concentrado à razão de 1% do peso corporal/dia. Foram avaliados a massa de forragem, os componentes botânicos do pasto e estruturais do CE e a taxa de lotação. Durante o período experimental, foram efetuados oito ciclos de pastejo. Sistemas forrageiros que envolvem gramíneas e leguminosas de diferentes ciclos proporcionam a utilização da área durante todo o ano agrícola em pastejo rotativo com bovinos leiteiros. Considerando-se a predominância das avaliações em cada pastejo, os sistemas forrageiros consorciados apresentam melhor resultado tanto paras as variáveis de massa de forragem quanto para a taxa de lotação.

The objective of this research was to evaluate three grazing systems (GS) with elephant grass (EG) + ryegrass (RG) + spontaneous growing species (SGS); EG + RG + SGS + forage peanut (FP); and EG + RG + SGS + red clover (RC), in order to use the area in rotational grazing during the agricultural year. EG was planted in rows with a distance of 4m between each row. In the cool-season, RG was sowed between EG rows and FP was preserved on GS. The experimental design was completely randomized with three treatments (GS), two replicates (paddocks) and independent evaluations (grazing cycles). For the evaluation lactating Holstein cows receiving 1% of BW/day feed supplement concentrate were used. The herbage mass, botanical composition of pasture, structural component of EG and stocking rate were evaluated. Eight grazing cycles were performed during the experimental period. Grazing systems involving grass and legume forage in different cycles provided the use of the area during the agricultural year in rotational grazing with dairy cattle. Considering the predominance of the evaluations in each grazing, the mixed grazing systems have better results for both forage mass and stocking rate variables.

Diaporthe: a genus of endophytic, saprobic and plant pathogenic fungi.

Diaporthe (Phomopsis) species have often been reported as plant pathogens, non-pathogenic endophytes or saprobes, commonly isolated from a wide range of hosts. The primary aim of the present study was to resolve the taxonomy and phylogeny of a large collection of Diaporthe species occurring on diverse hosts, either as pathogens, saprobes, or as harmless endophytes. In the present study we investigated 243 isolates using multilocus DNA sequence data. Analyses of the rDNA internal transcribed spacer (ITS1, 5.8S, ITS2) region, and partial translation elongation factor 1-alpha (TEF1), beta-tubulin (TUB), histone H3 (HIS) and calmodulin (CAL) genes resolved 95 clades. Fifteen new species are described, namely Diaporthe arengae, D. brasiliensis, D. endophytica, D. hongkongensis, D. inconspicua, D. infecunda, D. mayteni, D. neoarctii, D. oxe, D. paranensis, D. pseudomangiferae, D. pseudophoenicicola, D. raonikayaporum, D. schini and D. terebinthifolii. A further 14 new combinations are introduced in Diaporthe, and D. anacardii is epitypified. Although species of Diaporthe have in the past chiefly been distinguished based on host association, results of this study confirm several taxa to have wide host ranges, suggesting that they move freely among hosts, frequently co-colonising diseased or dead tissue. In contrast, some plant pathogenic and endophytic taxa appear to be strictly host specific. Given this diverse ecological behaviour among members of Diaporthe, future species descriptions lacking molecular data (at least ITS and HIS or TUB) should be strongly discouraged.

Características da ingestão de forragem por cordeiras nos estádios fenológicos da pastagem de azevém / Characteristics of forage intake of lambs at phenological stages of Italian ryegrass pasture

Foram estudadas as características do processo de ingestão de forragem por cordeiras em azevém (Lolium multiflorum Lam.) nos estádios fenológicos vegetativo, pré-florescimento e florescimento, por meio de testes de pastejo e da técnica da dupla pesagem. O delineamento experimental foi o inteiramente ao acaso, com três tratamentos e cinco repetições no estádio vegetativo e seis nos demais estádios. Os diferentes estádios fenológicos não influenciam a taxa de ingestão, a massa do bocado e a profundidade de bocado quando a oferta de forragem não é limitante ao consumo. O aumento na taxa de bocados é o mecanismo utilizado por cordeiras para manter a taxa de ingestão constante. Os modelos de regressão múltipla indicam que, dentre os atributos do pasto, sua altura é o de maior importância para determinar mudanças no comportamento ingestivo de cordeiras.

Utilizing grazing tests and the double weighing technique, the characteristics of the forage intake process of lambs grazing Italian Ryegrass pasture at vegetative, pre-flowering and flowering phenological stages were studied. A completely randomized design with three treatments and five replicates in the vegetative stage and six replicates in the other phenological stages was used. The intake rate, bite mass and bite depth were not influenced by the phenological stages when the intake was not limited by the forage allowance. By increasing bite rate lambs maintained a constant rate of intake. The multiple regression models indicate that among the herbage attributes, the sward height is the most important to determine changes in the grazing behavior of lambs on Italian Ryegrass pasture.

Genetic Diversity of Colletotrichum spp. an Endophytic Fungi in a Medicinal Plant, Brazilian Pepper Tree.

In this study, we reported thirty-nine endophytic fungi identified as Colletotrichum spp. associated with Brazilian pepper tree or aroeira (Schinus terebinthifolius Raddi. Anacardiaceae) in Paraná state, Brazil. These endophytes were identified by morphological and molecular methods, using PCR taxon-specific with CaInt/ITS4, CgInt/ITS4, and Col1/ITS4 primers, which amplify specific bands in C. acutatum, C. gloeosporioides lato sensu, and Colletotrichum boninensis, respectively, and by DNA sequence analysis of the nrDNA internal transcribed spacer region (ITS1, 5.8S, ITS2). We also assayed the presence of dsRNA particles in Colletotrichum spp. isolates. Combining both morphological characters and molecular data, we identified the species C. gloeosporioides, C. boninense, and C. simmondsii. However, we found a high genetic variability intraspecific in C. gloeosporioides which suggests the existence of several other species. Bands of double-stranded RNA (dsRNA) were detected in three of thirty-nine isolates. Identity of these bands was confirmed by RNAse, DNAse, and S1 nuclease treatments for the isolates LGMF633, LGMF726, and LGMF729. This is the first study reporting these particles of dsRNA in C. gloeosporioides.

Endophytic and pathogenic Phyllosticta species, with reference to those associated with Citrus Black Spot.

We investigated the identity and genetic diversity of more than 100 isolates belonging to Phyllosticta (teleomorph Guignardia), with particular emphasis on Phyllosticta citricarpa and Guignardia mangiferae s.l. occurring on Citrus. Phyllosticta citricarpa is the causal agent of Citrus Black Spot and is subject to phytosanitary legislation in the EU. This species is frequently confused with a taxon generally referred to as G. mangiferae, the presumed teleomorph of P. capitalensis, which is a non-pathogenic endophyte, commonly isolated from citrus leaves and fruits and a wide range of other hosts. DNA sequence analysis of the nrDNA internal transcribed spacer region (ITS1, 5.8S nrDNA, ITS2) and partial translation elongation factor 1-alpha (TEF1), actin and glyceraldehyde-3-phosphate dehydrogenase (GPDH) genes resolved nine clades correlating to seven known, and two apparently undescribed species. Phyllosticta citribraziliensis is newly described as an endophytic species occurring on Citrus in Brazil. An epitype is designated for P. citricarpa from material newly collected in Australia, which is distinct from P. citriasiana, presently only known on C. maxima from Asia. Phyllosticta bifrenariae is newly described for a species causing leaf and bulb spots on Bifrenaria harrisoniae (Orchidaceae) in Brazil. It is morphologically distinct from P. capitalensis, which was originally described from Stanhopea (Orchidaceae) in Brazil; an epitype is designated here. Guignardia mangiferae, which was originally described from Mangifera indica (Anacardiaceae) in India, is distinguished from the non-pathogenic endophyte, P. brazilianiae sp. nov., which is common on M. indica in Brazil. Furthermore, a combined phylogenetic tree revealed the P. capitalensis s.l. clade to be genetically distinct from the reference isolate of G. mangiferae. Several names are available for this clade, the oldest being P. capitalensis. These results suggest that endophytic, non-pathogenic isolates occurring on a wide host range would be more correctly referred to as P. capitalensis. However, more genes need to be analysed to fully resolve the morphological variation still observed within this clade.

Agrobacterium tumefaciens-mediated transformation of Guignardia citricarpa.

Guignardia citricarpa, the causal agent of Citrus Black Spot, was successfully transformed via Agrobacterium tumefaciens with cassettes for gfp and bar expression. Transformation is essential to understand the role of genes during interaction between plants and its pathogens. Using a binary plasmid vector based in the pPZP201BK, both germinated conidia and physically fragmented hyphae of G. citricarpa were transformed. Eight independent transformants of G. citricarpa resistant to ammonium glifosinate displayed GFP fluorescence. The majority (93.75%) of the G. citricarpa transformants was mitotically stable and contained a single T-DNA copy ectopically integrated to the chromosome. This is the first report of G. citricarpa transformation and will allow future work on virulence determinants of the fungus and possibly its control.

Fungicide resistance and genetic variability in plant pathogenic strains of Guignardia citricarpa / Resistência a fungicidas e variabilidade genética em linhagens patogênicas de plantas Guignardiacitricarpa

Citrus black spot (CBS) is a plant disease of worldwide occurrence, affecting crops in Africa, Oceania, and South America. In Brazil, climate provides favorable conditions and CBS has spread to the Southeast and South regions. CBS is caused by the fungus Guignardia citricarpa (anamorph: Phyllosticta citricarpa) and its control is based on the use of fungicides, such as benzimidazoles. In South Africa, the disease was kept under control for 10 years with benomyl, until cases of resistance to high concentrations of this fungicide were reported from all citrus-producing areas. Azoxystrobin (a strobilurin) has been found effective in controlling phytopathogens, including CBS, in a wide range of economically important crops. The present study investigated in vitro the effects of the fungicides benomyl and azoxystrobin on 10 strains of G. citricarpa isolated from lesions in citrus plants from Brazil and South Africa. Benomyl at 0.5 µg/mL inhibited mycelial growth in all strains except PC3C, of African origin, which exhibited resistance to concentrations of up to 100.0 µg/mL. The spontaneous mutation frequency for resistance to benomyl was 1.25 ï 10-7. Azoxystrobin, even at high concentrations, did not inhibit mycelial growth in any of the strains, but significantly reduced sporulation rates, by as much as 100%, at a concentration of 5.0 µg/mL. Variations in sensitivity across strains, particularly to the strobilurin azoxystrobin, are possibly related to genetic variability in G. citricarpa isolates.

A Mancha Preta dos Citros (MPC) tem ocorrência mundial afetando a produção de citros na África, Oceania e América do Sul. No Brasil, onde o clima é favorável ao seu desenvolvimento, a doença está espalhada nas regiões Sul e Sudeste. O controle da MPC, causada pelo fungo Guignardia citricarpa (anamorfo: Phyllosticta citricarpa) é baseado na aplicação de fungicidas, como os benzimidazóis. Na África do Sul, após 10 anos de controle da doença com o fungicida benomil, os casos de resistência a altas concentrações deste fungicida atingiram todas as áreas produtoras. O fungicida estrolilurina chamado azoxistrobina tem se mostrado eficiente no controle dos fitopatógenos de uma grande variedade de culturas economicamente importantes, incluindo a MPC. Neste trabalho foram investigados os efeitos in vitro dos fungicidas benomil e azoxistrobina em 10 linhagens de G. citricarpa isoladas de lesões em plantas cítricas no Brasil e na África do Sul. Houve inibição do crescimento micelial a 0,5 µg/mL do fungicida benomil entre as linhagens testadas, com exceção de PC3C de origem sul-africana, que apresentou resistência até a concentração de 100,0 µg/mL de benomil. A freqüência de mutação espontânea para resistência ao benomil foi de 1,25 ï 10-7. A estrobilurina azoxistrobina, mesmo em altas concentrações, não inibiu o crescimento micelial dos isolados, entretanto reduziu significativamente a produção de esporos, chegando a 100% de inibição em concentrações de 5,0 µg/mL de azoxistrobina. A variação na sensibilidade das linhagens, principalmente com a estrobilurina azoxistrobina, possivelmente está relacionada com a variabilidade genética dos isolados de G. citricarpa.

Fungicide resistance and genetic variability in plant pathogenic strains of Guignardia citricarpa.

Citrus black spot (CBS) is a plant disease of worldwide occurrence, affecting crops in Africa, Oceania, and South America. In Brazil, climate provides favorable conditions and CBS has spread to the Southeast and South regions. CBS is caused by the fungus Guignardia citricarpa (anamorph: Phyllosticta citricarpa) and its control is based on the use of fungicides, such as benzimidazoles. In South Africa, the disease was kept under control for 10 years with benomyl, until cases of resistance to high concentrations of this fungicide were reported from all citrus-producing areas. Azoxystrobin (a strobilurin) has been found effective in controlling phytopathogens, including CBS, in a wide range of economically important crops. The present study investigated in vitro the effects of the fungicides benomyl and azoxystrobin on 10 strains of G. citricarpa isolated from lesions in citrus plants from Brazil and South Africa. Benomyl at 0.5 µg/mL inhibited mycelial growth in all strains except PC3C, of African origin, which exhibited resistance to concentrations of up to 100.0 µg/mL. The spontaneous mutation frequency for resistance to benomyl was 1.25 × 10(-7). Azoxystrobin, even at high concentrations, did not inhibit mycelial growth in any of the strains, but significantly reduced sporulation rates, by as much as 100%, at a concentration of 5.0 µg/mL. Variations in sensitivity across strains, particularly to the strobilurin azoxystrobin, are possibly related to genetic variability in G. citricarpa isolates.