Viviana V. Rivera

Real-time PCR Quantification and Mycotoxin Production of Fusarium graminearum in Wheat Inoculated with Isolates Collected from Potato, Sugar Beet, and Wheat

ABSTRACT Fusarium graminearum causes Fusarium head blight (FHB) in small grains worldwide. Although primarily a pathogen of cereals, it also can infect noncereal crops such as potato and sugar beet in the United States. We used a real-time polymerase chain reaction (PCR) method based on intergenic sequences specific to the trichodiene synthase gene (Tri5) from F. graminearum. TaqMan probe and primers were designed and used to estimate DNA content of the pathogen (FgDNA) in the susceptible wheat cv. Grandin after inoculation with the 21 isolates of F. graminearum collected from potato, sugar beet, and wheat. The presence of nine mycotoxins was analyzed in the inoculated wheat heads by gas chromatography and mass spectrometry. All isolates contained the Tri5 gene and were virulent to cv. Grandin. Isolates of F. graminearum differed significantly in virulence (expressed as disease severity), FgDNA content, and mycotoxin accumulation. Potato isolates showed greater variability in producing different mycotoxins than sugar beet and wheat isolates. Correlation analysis showed a significant (P < 0.001) positive relationship between FgDNA content and FHB severity or deoxynivalenol (DON) production. Moreover, a significant (P < 0.001) positive correlation between FHB severity and DON content was observed. Our findings revealed that F. graminearum causing potato dry rot and sugar beet decay could be potential sources of inoculum for FHB epidemics in wheat. Real-time PCR assay provides sensitive and accurate quantification of F. graminearum in wheat and can be useful for monitoring the colonization of wheat grains by F. graminearum in controlled environments, and evaluating wheat germplasms for resistance to FHB.

Monitoring Fungicide Sensitivity of Cercospora beticola of Sugar Beet for Disease Management Decisions

Cercospora leaf spot, caused by the fungus Cercospora beticola Sacc., is the most serious and important foliar disease of sugar beet (Beta vulgaris L.) wherever it is grown worldwide. Cercospora leaf spot first caused economic damage in North Dakota and Minnesota in 1980, and the disease is now endemic. This is the largest production area for sugar beet in the United States, producing 5.5 to 6.0 million metric tons on approximately 300,000 ha, which is 56% of the sugar beet production in the United States. This Plant Disease feature article details a cooperative effort among the participants in the sugar beet industry in this growing area and represents a successful collaboration and team effort to confront and change a fungicide resistance crisis to a fungicide success program. As a case study of success for managing fungicide resistance, it will serve as an example to other pathogen-fungicide systems and provide inspiration and ideas for long-term disease management by fungicides.

Identification of the G143A mutation associated with QoI resistance in Cercospora beticola field isolates from Michigan, United States

BACKGROUND Cercospora leaf spot (CLS), caused by the fungus Cercospora beticola, is the most serious foliar disease of sugar beet (Beta vulgaris L.) worldwide. Disease control is mainly achieved by timely fungicide applications. In 2011, CLS control failures were reported in spite of application of quinone outside inhibitor (QoI) fungicide in several counties in Michigan, United States. The purpose of this study was to confirm the resistant phenotype and identify the molecular basis for QoI resistance of Michigan C. beticola isolates. RESULTS Isolates collected in Michigan in 1998 and 1999 that had no previous exposure to the QoI fungicides trifloxystrobin or pyraclostrobin exhibited QoI EC(50) values of ≤ 0.006 µg mL(-1) . In contrast, all isolates obtained in 2011 exhibited EC(50) values of > 0.92 µg mL(-1) to both fungicides and harbored a mutation in cytochrome b (cytb) that led to an amino acid exchange from glycine to alanine at position 143 (G143A) compared with baseline QoI-sensitive isolates. Microsatellite analysis of the isolates suggested that QoI resistance emerged independently in multiple genotypic backgrounds at multiple locations. A real-time PCR assay utilizing dual-labeled fluorogenic probes was developed to detect and differentiate QoI-resistant isolates harboring the G143A mutation from sensitive isolates. CONCLUSION The G143A mutation in cytb is associated with QoI resistance in C. beticola. Accurate monitoring of this mutation will be essential for fungicide resistance management in this pathosystem.

Temporal sensitivity ofAlternaria solani to foliar fungicides

Alternaria solani Sorauer (Ellis) is a causal agent of early blight of potato (Solanum tuberosum L.). Sensitivity to chlorothalonil, triphenyl tin hydroxide (TPTH), and mancozeb was determined for field isolatesof A. solani in 1998 and 1999. Specifically, the relative sensitivity ofA. solani to foliar fungicides in field populations collected from several commercial fields was evaluated over the course of two growing seasons. Sensitivity to these foliar fungicides was determined with anin vitro spore-germination assay method. The fungicide concentration that inhibited conidial germination by 50% (EC50) was estimated for each isolate. Sensitivity ofA. solani isolates to TPTH and mancozeb varied little during the growing season, possibly because there were few TPTH and mancozeb applications made to the fields studied. At several locations, however, repeated exposure ofA. solani populations to chlorothalonil resulted in considerable variability in sensitivity, frequently causing isolates to have decreased sensitivity to this fungicide at the end of the growing season. In five of seven fields, isolates of A.solani collected at the end of the season were significantly less sensitive to chlorothalonil than isolates collected at the beginning of the season.ResumenAlternaria solani Sorauer (Ellis) es el agente causal del tizon temprano de la papa (Solanum tuberosum L.). La sensibilidad al clorotalonil, trifenil hidroxido de estaño (TPTH) y mancozeb se determinó para aislamientos de campo deA. solani en 1998 y 1999. Específicamente se evaluó la sensibilidad relativa deA. solani a los fungicidas foliares en poblaciones de campo colectadas de varios campos comerciales en el transcurso de dos campanas de cultivo. La sensibilidad a estos fungicidas foliares fue determinada con un métodoin vitro de germinación de esporas. Para cada aislamiento se estimó la concentratión de fungicida que inhibió la germinación de las conidias en un 50% (EC50). La sensibilidad de los aislamientos deA. solani al TPTH y al mancozeb variaron muy poco durante el período de cultivo, posiblemente porque se hicieron pocas aplicaciones de estos productos a los campos estudiados. En varios lugares, sin embargo, la repetida exposición de las poblaciones deA. solani al clorotalonil resultaron en una considerable variabilidad en sensibilidad, frecuentemente causando que los alslamientos disminuyan su sensibilidad a este fungicida al final de cada estación de cultivo. En cinco de siete campos, los aislamientos deA. solani colectados al final del cultivo fueron significativamente menos sensibles al clorotalonil que los aislamientos colectados al comienzo de la época de cultivo.

Fungicide resistance assays for fungal plant pathogens.

Fungicide resistance assays are useful to determine if a fungal pathogen has developed resistance to a fungicide used to manage the disease it causes. Laboratory assays are used to determine loss of sensitivity, or resistance, to a fungicide and can explain fungicide failures and for developing successful fungicide recommendations in the field. Laboratory assays for fungicide resistance are conducted by measuring reductions in growth or spore germination of fungi in the presence of fungicide, or by molecular procedures. This chapter describes two techniques for measuring fungicide resistance, using the sugarbeet leaf spot fungus Cercospora beticola as a model for the protocol. Two procedures are described for fungicides from two different classes; growth reduction for triazole (sterol demethylation inhibitor; DMI) fungicides, and inhibition of spore germination for quinone outside inhibitor (QoI) fungicides.

First Report of a Novel Fusarium Species Causing Yellowing Decline of Sugar Beet in Minnesota

In the United States, yellows disease of sugar beet (Beta vulgaris), which causes wilt, early death, and yield reduction, is caused primarily by Fusarium oxysporum f. sp. betae (3,4), but F. graminearum (2) has also been implicated. During the past 3 years, a similar disease causing yellowing and severe decline appeared in some sugar beet fields of central and southwest Minnesota planted with cultivars resistant to yellows. The disease has become a concern to the local sugar beet industry, which produces 56% of sugar beets in the United States. From 2005 to 2007, isolations were made from sugar beets collected in commercial fields and from a Fusarium screening nursery showing symptoms of yellowing, interveinal chlorosis, scorching, stunting, vascular discoloration of the taproot, and early death of plants. Of 96 Fusarium isolates recovered and used in root-dip inoculation trials in the greenhouse, 58 were pathogenic to sugar beets. On the basis of morphology, 12 were identified as F. oxysporum, 6 as F. graminearum, and 40 as a novel Fusarium species. The remaining 38 isolates were nonpathogenic. All three pathogenic Fusarium species were isolated from taproots, but only the novel Fusarium was isolated from petioles. In culture, the novel Fusarium exhibited a bright orange color on the underside of potato dextrose agar medium and produced micro- and macroconidia sparsely. Hyphal tip isolates of all novel Fusarium isolates were pathogenic, causing typical yellowing symptoms and plant death to the Fusarium yellows susceptible sugar beet cv. VDH46177 in replicated greenhouse trials. Isolates were successfully reisolated from the symptomatic plants, fulfilling Kochs postulates. Restriction fragment length polymorphism (RFLP) endonuclease digestion patterns (Alu1, Fnu4HI, HaeIII, and HhaI) of the internal transcribed spacer (ITS) region of 40 pathogenic novel isolates showed a distinct pattern compared with known Fusarium species. Thin layer chromatography analysis of 13 novel isolates detected the type A trichothecenes neosolaniol and 4,15-diacetoxyscirpenol. Partial sequences of the translation elongation factor 1-α (TEF) from 12 single-spored novel Fusarium isolates were generated. BLAST analysis of the TEF sequence against the FUSARIUM-ID (1) and GenBank databases did not match any known Fusarium species. On the basis of pathogenicity, morphology, RFLP patterns, mycotoxin production, and TEF sequence analysis it appears that this is a new species of Fusarium, but additional multilocus phylogenetic analyses are warranted. The natural occurrence of this novel Fusarium pathogen in sugar beet may have implications in breeding for resistance to Fusarium yellows, since yellow decline has been observed in purportedly Fusarium-tolerant cultivars in the Minnesota and North Dakota production regions. References: (1) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (2) L. E. Hanson. Plant Dis. 90:686, 2006. (3). L. E. Hanson. Plant Dis. 90:1554, 2006. (4) C. E. Windels et al. Plant Dis. 89:341, 2005.

RESISTENCIA DE Phytophthora infestans (Montagne) de Bary A METALAXIL, EN CULTIVO DE PAPAS EN EL NORTE DE CHILE

Con el objeto de determinar la sensibilidad a metalaxil de dos poblaciones de Phytophthora infestans (Montagne) de Bary, en cultivos de papa (Solanum tuberosum L.) en diferentes localidades de la IV y V Region de Chile, se estudiaron mediante ensayos in vitro e in vivo 254 aislamientos, los cuales inicialmente fueron calificado como sensibles o resistentes de acuerdo a su crecimiento sobre agar centeno B y 10 mg L-1 de metalaxil. Su concentracion efectiva media (CE 50), fue estimada mediante un modelo de regresion lineal entre el logaritmo de seis concentraciones de metalaxil (0, 150, 250, 350, 450 y 550 mg L-1) y porcentaje de inhibicion transformado a unidades Probit. Los resultados de ambas temporadas demostraron que la totalidad de los aislamientos de P. infestans fueron resistentes a metalaxil. Aislamientos colectados en la IV Region durante 1999 presentaron variaciones de CE 50 entre 243 y 687 mg L-1 de metalaxil, 9,6% de ellos prresento CE 50 inferior a 300 mg L-1 de metalaxil. Aislamientos colectados en la V Region variaron su CE 50 entre 233 y 633 mg L-1 de metalaxil y solamente 10,1% de ellos presento CE 50 inferior a 300 mg L-1 de metalaxil. La totalidad de los aislamientos colectados en la temporada 2000 fueron resistentes al fungicida, 89,6 y 78,1% de los colectados en la IV y V Region respectivamente, presentaron CE 50 superiores a 350 mg L-1 de metalaxil. Los resultados del bioensayo fueron consistentes con el trabajo in vitro.

Comparative Pathogenicity and Virulence of Fusarium Species on Sugar Beet

In all, 98 isolates of three Fusarium spp. (18 Fusarium oxysporum, 30 F. graminearum, and 50 Fusarium sp. nov.) obtained from sugar beet in Minnesota were characterized for pathogenicity and virulence on sugar beet in the greenhouse by a bare-root inoculation method. Among the 98 isolates tested, 80% of isolates were pathogenic: 83% of the F. oxysporum isolates, 57% of the F. graminearum isolates, and 92% of the Fusarium sp. nov. isolates. Symptoms varied from slight to moderate wilting of the foliage, interveinal chlorosis and necrosis, and vascular discoloration of the taproot without any external root symptoms. Among the pathogenic isolates, 14% were highly virulent and 12% were moderately virulent. Most of the highly virulent isolates (91%) and moderately virulent isolates (89%) were Fusarium sp. nov. All pathogenic isolates of F. graminearum and most pathogenic isolates (87%) of F. oxysporum were less virulent. In general, more-virulent isolates induced first foliar symptoms earlier compared with less-virulent isolates. This study indicates that both F. oxysporum and Fusarium sp. nov. should be used in greenhouse and be present in field studies used for screening and developing sugar beet cultivars resistant to Fusarium yellows complex for Minnesota and North Dakota.

Cercospora beticola mating types in the North Central USA

Cereospora betkola the cause of leaf spot of sugar beet became a serious disease in 1981 and is endemic in several sugar beet producing areas of the US, including the Red River Valley of ND and MN. The disease reduces both yield and sucrose content of sugar beets. Control is by crop rotation, resistant varieties and timely fungicide applications. The fungicides used include triphenyl tin hydroxide (Super Tin, Agritin) , thiophanate methyl (Tops in) , tetraconazole (Eminent), pyraclostrobin (Headline), and trifloxystrobin (Gem), and are usually 2-4 applications are made the last half of season. Fungicide sensitivity of Cereospora betieola is a major concern because all fungicides used for C. betieola control have a history of resistance in sugarbeets or other crops