Real-Time Quantitative and Ion-Metal Indicator LAMP-Based Assays for Rapid Detection of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is one of the most devastating and cosmopolitan plant pathogens. Rapid detection of S. sclerotiorum can provide growers an advantage in knowing what control measures should be taken to minimize crop damage and financial losses caused by it. Loop-mediated isothermal amplification (LAMP) is a fast, sensitive, and specific nucleic acid amplification method that does not require a thermal cycler. This study aimed to develop a LAMP-based assay for the specific detection of S. sclerotiorum (Ss-LAMP). A real-time quantitative LAMP reaction (Ss-qLAMP) and a calcein ion indicator-based LAMP reaction (Ss-cLAMP) were designed, optimized, and tested on fungi, plant, and soil samples. The Ss-LAMP reactions were very specific and sensitive. Applying the artificially inoculated soil samples with DNA purified by five protocols in the Ss-qLAMP reaction, it was possible to detect and quantify the pathogen DNA, regardless of the extraction protocol. Naturally infected soybean tissues had the pathogen detected by Ss-cLAMP directly in the reaction tube with no DNA extraction requirement. The assays should be applicable for many types of samples, such as soil, spore traps, and plant tissues from several crops, with no requirement for DNA extraction. The Ss-LAMP reactions took less than 1 h to complete, and they can be made directly in the field with real-time quantitative results (Ss-qLAMP) or qualitative naked-eye visual results (Ss-cLAMP). Results were obtained with 10 pg of DNA or 10 ng of crude mycelium, suggesting a detection limit close to a single DNA copy. Ss-LAMP reactions will allow rapid and accurate diagnosis of S. sclerotiorum and assist in pathogen management and control.

Influência de herbicidas e fungicidas na germinação carpogênica de escleródios de Sclerotinia sclerotiorum (Lib. ) de Bary / Influence of herbicides and fungicides in the carpogenic germination of sclerotia of Sclerotinia sclerotiorum (Lib. ) de Bary

Sclerotinia sclerotiorum é um fungo polífago apresentando mais de 400 espécies hospedeiras. No Brasil relata-se que sua ocorrência tenha aumentado a partir da safra 2003/2004 em função do uso de sementes infectadas, falta de rotação de culturas, entre outras causas. Nos EUA, há relatos que o aumento da doença também tenha ocorrido devido à redução no uso de herbicidas do grupo das triazinas na cultura do milho, em rotação. Alguns trabalhos relatam que esse herbicida atua na formação de apotécios anormais e na inibição da produção de ascósporos. Frente a isso o presente trabalho objetivou o estudo do efeito de 12 herbicidas e 5 fungicidas sobre a germinação carpogênica de S. sclerotiorum. O trabalho foi conduzido em laboratório na Universidade Estadual de Ponta Grossa. Em caixas tipo gerbox foram colocados 80 gramas de substrato e 20 escleródios. A aplicação foi realizada utilizando um volume de calda de 400 L ha-1. Após a aplicação as caixas foram acondicionadas em ambiente controlado (20°C e fotoperíodo de 12 horas). Os tratamentos utilizados foram: glifosato, lactofen, atrazina, setoxidim, diuron, diuron + paraquat, imazaquim, trifluralina, diclofop metílico, clorimuron etílico, metribuzim e imazetapir (herbicidas) e fluazinam, procimidone, carbendazim, dimoxystrobin + boscalid e tiofanato metílico (fungicidas). Aos 36 dias após a aplicação foi realizada uma contagem do número de escleródios germinados, número de estipes e número de apotécios por escleródio. Para os dados de germinação de escleródios, foi constatado que somente dimoxystrobin + boscalid apresentou germinação inferior a 70%. Os demais tratamentos apresentaram germinação superior a 83%. Para número de estipes formadas por escleródio foi constatado que todos os tratamentos apresentaram de duas a quatro estipes por escleródio. Foi observado que os tratamentos com dimoxystrobin + boscalid, tiofanato metílico e carbendazim apresentaram as menores taxas de diferenciação de estipes em apotécios, ou seja, constatou-se a formação de estipes inviáveis. No tratamento com dimoxystrobin + boscalid foi constatado uma viabilidade de apenas 34,56% de suas estipes. Os resultados aqui apresentados mostram que a aplicação antecipada desses tratamentos pode ser realizada visando atingir a fonte de inóculo (escleródios) no solo, reduzindo o potencial de infecção dos mesmos e consequentemente sua incidência sobre culturas.

Sclerotinia sclerotiorum is a pathogenic fungus with over 400 host species. In Brazil, it is reported that its occurrence has increased since the 2003-2004 growing season due to use of infected seed, lack of crop rotation, and several other factors. In the United States, reports suggest that the disease increase has occurred due to reduced use of herbicides from the triazine group on corn in rotation with soybean. Reports show that these herbicides affect S. sclerotiorum, causing the formation of abnormal apothecia and inhibiting ascospore production. Because of the possible linkage with herbicide use, a study was designed to examine the effect of 12 herbicides and 5 fungicides on the carpogenic germination of S. sclerotiorum. This work was performed at Universidade Estadual de Ponta Grossa. Eighty grams of sterilized field soil and 20 sclerotia, placed on the soil surface, were added to "gerbox" boxes. Chemicals were applied once using a rate of 400 L ha-1. After application, boxes were incubated in a controlled environment (20°C with a photoperiod of 12 hours). The treatments used were: glyphosate, lactofen, atrazine, sethoxydim, diuron, diuron + paraquat, imazaquin, trifluralin, diclofop methyl, chrlorimuron ethyl, metribuzin and imazethapyr (herbicides) and fluazinam, procymidone, carbendazim, dimoxystrobin + boscalid and thiophanate methyl (fungicides). The number of germinated sclerotia, stipes and apothecia was recorded 36 days after chemical application. For sclerotia germination, only dimoxystrobin + boscalid reduced germination below 70%. All other treatments had greater than 83% sclerotia germination. The number of stipes produced per sclerotium was constant for all treatments (two to four stipes per sclerotium). Dimoxystrobin + boscalid, thiophanate methyl and carbendazim had the lowest level of apothecia formation, which means inviable stipes were formed. For the treatment of dimoxystrobin + boscalid, only 34.56% of stipes developed into mature apothecia. The results of this study demonstrate that application of herbicides did not affect the sclerotia of S. sclerotiorum. Three of the five fungicide tested in this trial, dimoxystrobin + boscalid, thiophanate methyl and carbendazim, can reduce inoculum on the soil, which, in turn, would likely reduce disease incidence in the following crop.