The fungus <i>Colletotrichum truncatum</i> is a potential biocontrol agent (BA) against the noxious weed scentless chamomile (<i>Metricaria perforata</i> Mérat; syn.: <i>Tripleurospermum perforatum</i> (Mérat) Lainz) in western Canada. This potential BA, however, is taxonomically related to the anthracnose pathogen on lentil, raising questions about crop safety. Ribosomal DNA (rDNA) internal transcribed space (ITS) regions of <i>C. truncatum</i> isolates collected from different plant hosts were examined, and compared with additional Colletotrichum species. Sequences were amplified with the universal primers its4 and its5, and <i>C. truncatum</i> isolates from scentless chamomile and selected legume crops were differentiated consistently. All scentless chamomile isolates fell within a single cluster in phylogenetic trees, regardless of their geographic origins. These isolates were more closely related to lentil isolates of <i>C. truncatum</i> than to isolates from the other host species. Soybean isolates, with more falcate and slender conidia and slightly bigger appressoria, were distinguishable from other <i>C. truncatum</i> isolates, while the isolates from scentless chamomile, lentil and pea were morphologically more similar. Based on sequence information, strain-specific PCR primers were designed for <i>C. truncatum</i> isolates from these hosts and used to amplify specific DNA bands (markers) from isolates of <i>C. truncatum</i>. This technique may be used for rapid detection and differentiation of <i>C. truncatum,</i> from scentless chamomile and designated legume species, as well as for tracking the BA after release. Inoculation trials were conducted using detached leaves and whole plants to determine potential cross infection of these <i>C. truncatum</i> isolates. Isolates from scentless chamomile caused disease only on their original host, but not on lentil, pea, soybean or alfalfa. In contrast, lentil isolates caused severe disease on lentil and pea, light symptoms on alfalfa, but no disease on the other hosts tested. Potential penetration of lentil leaves by scentless chamomile isolates was tested, with 2-23% incidence of the fungus from inoculated detached, senescence leaves but disease symptoms were not observed on either detached leaves or whole plants. Examination of the infection process revealed that scentless chamomile and lentil isolates had a similar pattern of infection and disease development on their respective hosts; infection vesicles were produced 24 h after inoculation, both primary and secondary infection hyphae were present, and the onset of disease symptoms tended to coincide with the development of secondary hyphae. The current study provided molecular and pathological evidence that differentiates the potential BA of scentless chamomile from <i>C. truncatum</i> isolates from lentil, pea and soybean.

The fungus <i>Colletotrichum truncatum</i> is a potential biocontrol agent (BA) against the noxious weed scentless chamomile (<i>Metricaria perforata</i> Mérat; syn.: <i>Tripleurospermum perforatum</i> (Mérat) Lainz) in western Canada. This potential BA, however, is taxonomically related to the anthracnose pathogen on lentil, raising questions about crop safety. Ribosomal DNA (rDNA) internal transcribed space (ITS) regions of <i>C. truncatum</i> isolates collected from different plant hosts were examined, and compared with additional Colletotrichum species. Sequences were amplified with the universal primers its4 and its5, and <i>C. truncatum</i> isolates from scentless chamomile and selected legume crops were differentiated consistently. All scentless chamomile isolates fell within a single cluster in phylogenetic trees, regardless of their geographic origins. These isolates were more closely related to lentil isolates of <i>C. truncatum</i> than to isolates from the other host species. Soybean isolates, with more falcate and slender conidia and slightly bigger appressoria, were distinguishable from other <i>C. truncatum</i> isolates, while the isolates from scentless chamomile, lentil and pea were morphologically more similar. Based on sequence information, strain-specific PCR primers were designed for <i>C. truncatum</i> isolates from these hosts and used to amplify specific DNA bands (markers) from isolates of <i>C. truncatum</i>. This technique may be used for rapid detection and differentiation of <i>C. truncatum,</i> from scentless chamomile and designated legume species, as well as for tracking the BA after release. Inoculation trials were conducted using detached leaves and whole plants to determine potential cross infection of these <i>C. truncatum</i> isolates. Isolates from scentless chamomile caused disease only on their original host, but not on lentil, pea, soybean or alfalfa. In contrast, lentil isolates caused severe disease on lentil and pea, light symptoms on alfalfa, but no disease on the other hosts tested. Potential penetration of lentil leaves by scentless chamomile isolates was tested, with 2-23% incidence of the fungus from inoculated detached, senescence leaves but disease symptoms were not observed on either detached leaves or whole plants. Examination of the infection process revealed that scentless chamomile and lentil isolates had a similar pattern of infection and disease development on their respective hosts; infection vesicles were produced 24 h after inoculation, both primary and secondary infection hyphae were present, and the onset of disease symptoms tended to coincide with the development of secondary hyphae. The current study provided molecular and pathological evidence that differentiates the potential BA of scentless chamomile from <i>C. truncatum</i> isolates from lentil, pea and soybean.

A cultura da soja tem grande importância na segurança alimentar mundial, sendo o Brasil o segundo maior produtor do grão. Dentre os fatores que afetam a produção da cultura, as doenças se destacam e uma das principais é a antracnose. Diferentes espécies de Colletotrichum têm sido relacionadas à antracnose da soja, como C. truncatum, geralmente reportado como o principal agente causal da doença, e o C. plurivorum, anteriormente nomeado C. cliviae, uma espécie recentemente descrita e associada ao patossistema. Desta forma, objetivou-se caracterizar de forma filogenética, morfológica e cultural a espécie C. plurivorum comparando-a com C. truncatum; comparar a agressividade e a capacidade de transmissão semente-planta e planta-semente entre C. plurivorum e C. truncatum na cultura da soja; testar a reação de diferentes cultivares de soja a C. plurivorum; e verificar a sensibilidade in vitro de C. plurivorum a fungicidas registrados no Brasil para controle da doença. Baseado na análise filogenética Bayesiana, os isolados anteriormente chamados de C. cliviae foram reclassificados como C. plurivorum, agrupando-se juntamente com novos isolados de coletas recentes. Um isolado foi classificado como C. musicola, primeiro relato desta espécie isolada de planta sintomática de soja. Os isolados de C. plurivorum apresentaram taxas de crescimento micelial maior do que os de C. truncatum, enquanto que a esporulação dos isolados da segunda espécie foi superior aos da primeira. No teste de comparação de agressividade, o isolado de C. truncatum se mostrou mais agressivo do que os isolados de C. plurivorum à cultivar AMS Tibagi, após inoculações com suspensões de esporos no estágio de crescimento V2-V3. No teste de transmissão semente-planta, as taxas de transmissão de C. plurivorum e de C. truncatum da semente para a planta variaram entre 37% e 79%. Não foi observada transmissão dos patógenos para os grãos em teste de sanidade quando as plantas foram inoculadas nos estágios R2 e R5, além de não afetarem a produção de vagens e grãos. No teste de reação de cultivares de soja a C. plurivorum, a AMS Tibagi foi a que apresentou maior resistência, enquanto que as demais cultivares avaliadas foram afetadas significativamente nos parâmetros de qualidade fisiológica avaliados. Nas avaliações de sensibilidade a fungicidas, isolados de C. plurivorum e C. musicola, apresentaram insensibilidade, baixa ou média sensibilidade aos fungicidas trifloxistrobina e piraclostrobina. Para esses mesmos fungicidas, os isolados de C. truncatum e C. sojae foram classificados como altamente sensíveis (CE50 menor que 1 &#181;g mL-1). Os isolados de C. plurivorum e C. musicola apresentaram a mutação E198A no gene da beta-tubulina, que confere resistência ao tiofanato-metílico, enquanto que os isolados de C. truncatum foram considerados altamente sensíveis ao produto e não apresentaram a mutação no gene analisado. Isolados de C. plurivorum e de C. truncatum, avaliados no teste de inibição do crescimento micelial, foram considerados altamente sensíveis aos fungicidas difenoconazol e fludioxonil. Identificar e conhecer as características das espécies Colletotrichum que afetam uma das principais commodities do Brasil é fundamental para entender a epidemiologia da doença e obter sucesso no desenvolvimento de estratégias de controle mais eficientes. / Soybean cultivation has great importance in world food security with Brazil being the second largest grain producer. Among the factors that affect crop production, diseases stand out and one of the main ones is anthracnose. Different species of Colletotrichum have been related to soybean anthracnose, such as C. truncatum, generally reported as the main causal agent of the disease, and C. plurivorum, previously named C. cliviae and a recently described pathosystem associated species. The aim of this work was to characterize in a phylogenetic, morphological and cultural way the species C. plurivorum comparing it with C. truncatum; to compare the aggressiveness and seed-plant and plant-seed transmission capacity between C. plurivorum and C. truncatum in soybean cultivation; to test the reaction of different soybean cultivars to C. plurivorum; and to verify C. plurivorum in vitro sensitivity to fungicides registered in Brazil for disease control. Based on Bayesian phylogenetic analysis, the strains previously called C. cliviae were reclassified as C. plurivorum, grouped together with new strains from recent collections. One strain was classified as C. musicola, the first report of this species on soybean symptomatic plant. The C. plurivorum strains had higher mycelial growth rates than those of C. truncatum, whereas the sporulation of the second species was superior. C. truncatum was more aggressive than C. plurivorum to the AMS Tibagi cultivar after inoculations with spore suspensions at the V2-V3 growth stage. In the seed-plant transmission test, C. plurivorum and C. truncatum transmission rates from the seed to the plant varied between 37% and 79%. Transmission of the pathogens to the grains was not observed when the plants were inoculated in stages R2 and R5, besides not affecting the production of pods and grains. Among the tested cultivars, \'AMS Tibagi\'showed the highest resistance to C. plurivorum, while the other cultivars were significantly affected in the physiological quality parameters. C. plurivorum and C. musicola strains showed insensitivity, low or medium sensitivity to trifloxystrobin and pyraclostrobin fungicides. For these same fungicides, C. truncatum and C. sojae strains were classified as highly sensitive (EC50 less than 1 &#181;g mL-1). C. plurivorum and C. musicola strains showed the E198A mutation in the beta-tubulin gene, which confers resistance to thiophanate-methyl, whereas C. truncatum strains were considered highly sensitive to this fungicide and did not present the mutation in the beta-tubulin gene. C. plurivorum and C. truncatum strains were considered highly sensitive to difenoconazol and fludioxonil fungicides. Identifying and knowing the characteristics of the Colletotrichum species that affect one of the main commodities in Brazil is fundamental to understand the epidemiology of the disease and to succeed in the development of more efficient control strategies.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2016-04-26T11:31:40Z No. of bitstreams: 1 texto completo.pdf: 828935 bytes, checksum: 1db7e181e01f2e3911c0020c68d4f833 (MD5) / Made available in DSpace on 2016-04-26T11:31:40Z (GMT). No. of bitstreams: 1 texto completo.pdf: 828935 bytes, checksum: 1db7e181e01f2e3911c0020c68d4f833 (MD5) Previous issue date: 2015-02-19 / Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A Anthracnose, causada pelo fungo Colletotrichum truncatum, é uma das doenças de soja mais importantes no mundo, mas não há estudos avaliando as alterações fisiológicas neste patossistema. Portanto, uma abordagem para avaliar os eventos que ocorrem no local da infecção e perto da área infectada na folha, ao longo do tempo, contribuirá para uma melhor compreensão da interação planta-hospedeiro e atividade fotossintética. Assim, o presente estudo buscou investigar parâmetros de fluorescência da clorofila a (Chl a) na área da lesão e uma área adjacente, associando os ás trocas gasosas e avaliação de pigmentos fotossintéticos em plantas de soja inoculadas ou não inoculadas com C. truncatum. O parâmetros de trocas gasosas não foram alterados em plantas inoculadas. No entanto, ocorreu redução da concentração de Chl a, Ch b e da Chl total (a + b) nas plantas inoculadas as 72 e 144 horas após a inoculação (hai), com redução máxima à 144 hai de 24% para Chl a, que demonstrou ser mais sensível que a Chl b, ocasionando, portanto, a redução da razão Chl a/ Chl b. Também foi encontrado queda em valores de fluorescência da clorofila a como, Fluorescência inicial (Fo), Fluorescência máxima (Fm), Eficiencia quântica máxima do fotossistema II (Fv/Fm), Rendimento quântico de dissipação de energia regulada Y(NPQ) e coeficiente não- fotoquímico (qN), e um acréscimo no Rendimento quântico efetivo do PSII Y(II), Rendimento quântico de dissipação de energia não regulada (NO) e coeficiente fotoquímico (qP) nas área sintomática de plantas inoculadas. Entretanto esses parâmetros sofreram pequenas alterações nas áreas adjacentes das plantas inoculadas, diferindo apenas em alguns tempos. Demonstrando, dessa forma, um menor efeito do patógeno nas áreas adjacentes. / Anthracnose, caused by Colletotrichum truncatum, is one of the most important soybean diseases worldwide. However, there are no studies evaluating the physiological changes affecting this pathossystem. Therefore, one approach to evaluating events that occur at the site of infection and near the infected area on the leaf, over time, will contribute to a better understanding of the host-plant interaction and photosynthetic activity. The present study aimed to investigate chlorophyll a fluorescence parameters at injured and adjacent areas and the related changes in gas exchange and evaluation of photosynthetic pigments in soybean plants inoculated or non-inoculated with C. truncatum. There were no significant differences regarding gas exchange parameters for inoculated plants. However, there was a reduction in the concentration of Chl a, Chl b e Chl total (a + b) of inoculated plants in the 72 and 144 hours after inoculation (hai). Reduction in chlorophyll a fluorescence parameters to as initial fluorescence (Fo), maximal fluorescence (Fm), maximal photosystem II quantum yield (Fv/ Fm), quantum yield of regulated energy dissipation Y (NPQ) and coefficient non-photochemical (qN), and an increase in the Effective PSII quantum yield Y (II), quantum yield of non- regulated energy dissipation (NO) and photochemical coefficient (qP) in the symptomatic area plants inoculated. However, these parameters have undergone minor adjacent areas of inoculated plants, differing only in a few days. Demonstrating a smaller effect of the pathogen in adjacent.