Plant Pathology

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Research from the Department of Plant Pathology. The department website may be found at https://www.ag.ndsu.edu/plantpath

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Scanning Electron Microscope Examination of Sugarbeet Flowers and Fruits Infected with Phoma Betae
(North Dakota State University, 1980) El-Nashaar, Hossien Mahmoud
There are three natural openings in a mature sugarbeet fruit which serve as avenues of entry for microorganisms: 1) the basal pore which contains dried parenchyma and vascular tissue and is the point where the flower was connected to the stalk; 2) the apical pore where the style was inserted; and 3) the peripheral zone of dehiscence where the operculum separates from the fruit cavity wall during germination. The apical pore was first described in this study. Scanning electron microscopy of the naturally infected fruits showed, for the first time, hyphal penetration through both the basal pore and the peripheral zone. Examination of sugarbeet flowers artificially infected with Phoma betae also showed fungal penetration through the apical pore. Dense hyphal growth was associated with stigmal lobes and ungerminated pollen grains. Fungal growth apparently was stimulated by excretions from the stigma. Penetration of the fruit cavity wall and the operculum would render the fungus inaccessible to protectant fungicides and explains why the most successful seed treatments for P. betae have included volatile mercury fungicides or seed soak in thiram. Such treatment allows direct contact between the toxin and the pathogen.
Pathogenic Variation, Host Specialization and Aggressiveness in Helminthosporium Sativum
(North Dakota State University, 1984) El-Nashaar, Hossien Mahmoud
Common root rot, caused by Cochliobolus sativus, is an important disease of spring wheat and barley in the northern Great Plains. While the disease has been widely studied, extensive systematic investigation of pathological variation in C. sativus populations has not been reported. Five widely separated North Dakota counties were selected for field sampling. More than 430 isolates of C. sativus were obtained from wheat and barley plants. Pathogenicity of isolates was assessed according to amount of disease on wheat test plants in randomized, replicated greenhouse trials. Data were analyzed using the UNIVARIATE procedure of the SAS computer package. Relative pathogenicities of isolates were normally distributed continuous variables. Although the range of pathogenicity in populations of C. sativus within counties was similar, the means and variances were slightly different. Repeated planting of the same crop often results in an increase of inoculum of soilborne plant pathogens, but little is known of the effect on pathogenicity in the pathogen population. I collected C. sativus from symptomatic wheat plants from a plot planted continuously to wheat for over 90 years and from normally rotated grain fields in the surrounding area. Relative pathogenicity of isolates was tested in replicated greenhouse trials using wheat plants scored for disease six weeks after inoculation. Isolates from the continuous wheat plot were more pathogenic on average than isolates from commercial fields. Long term continuous cropping to wheat appears to shift the population of C. sativus toward more aggressive types, although many less pathogenic types also remain. Variability in C. sativus has been recognized in morphology of culture and pathogenicity for many years. Variability on virulence and/or aggressiveness of C. sativus isolates is not well documented. From the original collection of 432 isolates, 50 were selected to cover the entire range of pathogenicity. In three experiments, relative pathogenicity of the isolates was evaluated according to the amount of disease on each of four durum wheat cultivars. Aggressiveness patterns in the C. sativus-wheat system were reasonably reproducible. The C. sativus isolates originally selected for differential responses to wheat cultivars were not consistent in repeated testing. It was concluded that a range of pathogenicity (= aggressiveness ) exists in the C. sativus population and the apparent host differential responses are not repeatable and are probably experimental artifacts.
The Impact of Plant Age, Fungicide Application Methodology and Timing, and Depth of Soil Inoculation on Infection by Rhizoctonia solani on Sugarbeet.
(North Dakota State University, 2010) Pooran-DeSouza, Scmwattie
Rhizoctonia root rot caused by Rhizoctonia solani (Kuhn), is the most important problem faced by sugarbeet (Beta vulgaris L.) grm.vers in North Dakota and Minnesota. Research was conducted that may be used to manage the disease. Six cultivars from 2 to 8- leaf stage were evaluated for their ability to withstand infection after soil inoculation by R. solani AG 2-2 IIIB. All leaf stages of the cultivars evaluated were susceptible to R. solani. Sugarbeet plants at the 2-leaf stages were most susceptible and had significantly higher root rot severity than plants at the 4, 6 and 8-leaf stages. Cultivars Crystal 454 and Hillshog 3035 had the lowest root rot severity of the cultivars evaluated, but root rot severity was still greater than 50%. The cultural practice of planting early in soils when the temperature is about 10°C at the 10 cm depth may result in plants being older and more tolerant by the time the pathogen becomes infective at warmer soil temperatures. However, the fact that all plant stages were susceptible to R. solani may require additional protective measures in highly infested fields with a knovm history of severe Rhizoctonia root rot. During greenhouse research, it was observed that azoxystrobin fungicide applied as a hypocotyl drench provided excellent control of Rhizoctonia root rot. It is recommended that growers use a foliar banded application of azoxystrobin at the 4-leaf stage to control Rhizoctonia root rot. Research was conducted to compare and evaluate the effect of foliar band and hypocotyl drench applications of azoxystrobin to control R. solani. The study showed that foliar banded and hypocotyl drench applications of azoxystrobin provided significantly similar disease control under conditions that were ideal for disease development However, disease from plants with a hypocotyl drench application was not significantly different than the non-inoculated control suggesting that further testing should be done to determine the utility of this application methodology in field conditions. Research was conducted to determine the best time to apply azoxystrobin fungicide for effective R. solani control relative to timing of soil inoculation. Sugarbeet hypocotyls were drenched at the 4-leaf stage at 0, 7, 14, 21, and 28 days pre-inoculation and at 0, 3, 10, 14, and 21 days post-inoculation. Azoxystrobin applications prior to inoculation resulted in significantly lower root rot compared to fungicide applications at post inoculation. Among the post inoculation applications, treatments where the fungicide was applied within 2 hours provided the best disease control. Fungicide application at pre-inoculation provided effective control at all timings evaluated: This research reinforces the need for azoxystrobin application before infection to control the disease in field conditions. The depth at which R. solani caused root rot infection of sugarbeet was studied after burying R. solani AG 2-2 IIIB inoculurn at depths of 2.54, 7 .62, and 12. 7 cm. R. solani AG 2-2 IIIB infections occurred at all depths of inoculation. However, inoculum buried at 2.54 cm depth had significantly higher root rot severity than inoculum buried deeper. Root rot symptoms were prevalent on the upper portion of the sugarbeet root just below the soil line irrespective of the depth of inoculurn placement. This suggests that the upper part of the root below the soil line is most vulnerable to R. solani infection. Consequently, in the soil fungicide application should target the root area just below the soil line for effective disease control.
Studies on the Population Biology of Colletotrichum coccodes Using AFLP and SCAR Markers
(North Dakota State University, 2010) Alananbeh, Kholoud Mohammad
Black dot is a blemish disease of potato caused by the fungus Colletotrichum coccodes (Wallr.) Hughes. with worldwide distribution. It occurs in Africa, Asia, Australia, Europe, North America, South America, and Central America. C. coccodes has no known sexual cycle, but genetic exchange is possible through vegetative conjugation. The determination of VCG among fungal isolates is usually based on the complementation tests with nitrate nonutilizing (nit) mutants which enables researchers to compare strains of pathogens. Eight VCGs have been identified for the Europe/Israel population, seven for the North American population, and six for the Australian population. Variation in aggressiveness, morphological traits, and physiological traits have been detected among the different C. coccodes VCGs. AFLP markers have been used also to study relationships within and among North American VCGs. With this method, five VCGs were separated from C. coccodes isolates, coinciding almost completely with the six VCGs of nit mutants. Additionally, a relationship of specific AFLP bands to corresponding VCGs was reported using AFLP analysis. The objectives of this study were: to develop VCG-specific sequence characterized amplified regions (SCAR) markers, to study the population biology of C. coccodes of North America using the AFLP method, and to study genetic diversity of the global populations of C. coccodes. A total of 88 isolates representing the different C. coccodes VCGs were analyzed via the AFLP method to generate SCAR markers. A total of 47 primers were designed and evaluated in PCR reactions. Only one primer, AGb6F/R, with 156 bp amplification PCR product was found to be specific for NA-VCG6 and NA V CG 7 of C. coccodes. For the second objective, 210 loci were generated and used to cluster the isolates into their NA-VCGs and to test the genetic structure of the North American population of C. coccodes. C. coccodes isolates recovered from potato plants were assigned to fourNA-VCGs: NA-VCGl, NA-VCG2, NA-VCG4/5, and NA-VCG6/7. No isolates tested belonged to NA-VCG3. NA-VCG2 was the dominant group in the population (n=238) and was the most frequently detected NA-VCG among states, fields, farms, and plants. However, in several instances there was more than one NA-VCG recovered from the same plant, field, farm, and state, indicating variability within the C. coccodes population in United States. Genetic differentiation among the nine states was 0.331, and overall gene flow for the 366 isolates was (Nm =1.01). For the genetic diversity of the global C. coccodes population, 855 isolates were studied. Three primer pairs were used and generated 210 loci. Based on this study, the population of C. coccodes exists as one large population with four main groups (NA-VCGl/3; NA-VCG2; NA-VCG4/5; and NA-VCG6/7). NA-VCG5 was the most common VCG globally followed by NA-VCG2. Among the five regions studied, there was relatively low gene diversity (0.222). The overall gene flow (Nm) was 1.25, meaning that one or more individuals are exchanged among the five regions each generation and that the populations will gradually become similar. Most of the variation among the five geographic regions originated from within population differentiation. Among VCGs of C. coccodes, there was high VCG differentiation (GsT=0.463), meaning there is a differentiation among the different C. coccodes VCGs and a population structure exists. AFLP analysis proved to be valuable in differentiating and studying the global population of C. coccodes.
Association Mapping of Resistance to Common Root Rot and Spot Blotch in Barley, and Population Genetics of Coch/iobolus sativus
(North Dakota State University, 2010) Gyawali, Sanjaya
Cochliobolus sativus (Ito & Kurib.) Drechsl. ex Dast. [anamorph, Bipolaris sorokiniana (Sacc. in Sorok.) Shoem.] is an important fungal pathogen, which causes common root rot (CRR), spot blotch (SB) and black point/kernel blight in barley in North America. Use of genetic resistance has been effective against SB presumably due to presence of durable resistance in North America. However, recently emerged virulence groups have overcome durable resistance in barley. Additionally, the genetics of resistance to CRR is poorly understood. Therefore, the objectives of current studies are multifaceted. To identify the sources of resistance to CRR, 824 contemporary barley lines from the USDA-CSREES Barley Coordinated Agricultural Project (CAP) were evaluated for resistance to CRR under natural inoculum pressure in the field during 2006 - 2008. Additionally, resistance to CRR (n = 384 lines) and SB (n = 386 lines) was also evaluated in the greenhouse. The results indicate that only 0.9% of breeding lines showed resistance to CRR in field experiments during 2006-2008. None of the genotypes showed resistance to virulent isolates in greenhouse experiments. Hordeum jubatum sp. jubatum (Accession # CGN 13044) showed 12% CRR severity against highly virulent isolates. Therefore, this accession can be used as a potential source of resistance to CRR in the future. In the SB experiment, only 0.5% of the 386 genotypes showed resistant responses to isolate 4008. The barley line NDB112 with durable resistance to SB also showed a highly susceptible reaction to this isolate. To map QTL for CRR and SB resistance, association mapping was employed using the CAP06 population (n = 384), CAP07 population (n = 384) for CRR resistance and the CAP06 population (n = 384) for SB resistance. In all association analyses, 3072 single nucleotide polymorphism (SNP) markers were used. The results suggest five QTL resistance to CRR, CRR-3H-28-51, CRR-SH- 180-195, CRR-6H-30-64, CRR-6H-91-97, and CRR-lH-50-86 were detected in chromosomes 3H, 5H, 6H, and 7H. Two QTL resistance to spot blotch, Rcs-1H- 84.6 and Rcs-2H-106-122 were identified in chromosomes 1 Hand 2H, respectively. These QTL didn't coincide with any of the QTL reported earlier and confer resistance to virulence group 7.7.3.6. To investigate the virulence spectrum of C. sativus isolates collected from North Dakota (ND), 12 barley genotypes were inoculated with 12 virulent C. sativus isolates on both root and leaf. The results suggest that different virulence groups for CRR and SB diseases exist in the pathogen population. To understand the population structure of C. sativus populations collected from Australia and the USA (ND), 289 single-spore isolates were analyzed for amplified fragment length polymorphism (AFLP) using three AFLP primer combinations. Moderate to high gene diversity (H = 0.27 - 0.35) and high genotypic diversity (GD= 1) within C. sativus populations indicate occurrence of genetic recombination other than sexual in C. sativus populations. The moderate to high population differentiation (Gst = 0.196), moderate multilocus linkage disequilibrium (rd = 0.046 - 0.118), and low gene flow (Nm= 2.0) suggest the occurrence of different populations of C. sativus in the field.
Screening of Germplasm Accessions from the Brassica Species for Resistance against PG3 and PG4 Isolates of Blackleg
(North Dakota State University, 2011) Marino, Dante
Blackleg is a disease of canola and rapeseed cultivars that is caused by the fungus Leptosphaeria maculans (Desm.) Ces. & de Not., and it is by far the most destructive pathogen of canola in North America. In recent years, blackleg strains belonging to pathogenicity groups (PG) 3 and 4 have been discovered in North Dakota. Recent outbreaks of the disease have added a sense of urgency to characterize the risk these new strains represent for the canola industry and to identify sources of resistance against them. Thus, the objectives of this study were to screen germplasm collections of Brassica rapa, B. napus. and B. juncea for their reaction to PG3 and PG4 and to evaluate the reaction of a sample of currently used canola commercial cultivars grown in North Dakota to PG3 and PG4 as means to estimate the risk these new strains represent. All canola germplasm and commercial cultivars were evaluated in replicated trials in greenhouse conditions using cotyledon bioassays. In 2009 and 2010, the effect of these strains, using five inoculation sequences, on the reaction of canola seedlings was also evaluated. Field trials were not conducted because of the limited geographical distribution of the new strains. No adequate sources of resistance were identified among the 277 B. rapa and 130 B. napus accessions evaluated; however, 22 of the 406 accessions of Brassicajuncea evaluated were considered to have moderate levels of resistance. B. juncea seedlings that survived these inoculations were self-pollinated and their progeny (F1) were also screened. As before, surviving seedlings were self-pollinated. These F2 seeds are the elite materials that could be used in future breeding programs. The complementary study evaluating the role of sequence inoculations in reaction of canola seedlings to blackleg indicated that an increased susceptibility to PG3 occurred when seedlings were first inoculated with PG4; however, reaction to PG4 was not enhanced by a prior inoculation with PG3. All 75 commercial cultivars evaluated were susceptible to PG3 and PG4, indicating that the risk these new strains represent to the canola industry of the region is serious. Further, when a subsample of 16 cultivars were challenged with PG2, they were either resistant or moderately resistant, suggesting the ratings the industry are using relate to reaction of those cultivars to PG2 but not to the new strains; thus, growers should use caution when using these ratings while deciding on which cultivars to plant.
Prevalence of Pathogenicity Groups of the Blackleg Species Complex on Canola in North Dakota
(North Dakota State University, 2011) Mazurek, Shanna Andrea
North Dakota is the leading producer of canola (Brassica napus) in the United States. Blackleg, caused by two species of Leptosphaeria, L. maculans (Desm.) Ces & De Not and L. biglobosa, is an economically important disease wherever canola is grown. The virulence profile of these species has been classified in six pathogenicity groups (PG) based on inoculations on three differential cultivars, 'Westar', 'Glacier', and 'Quinta'. A PG-1 reaction is caused only by L. biglobosa isolates. By 2001, PG-2 was the predominant profile in North Dakota, although PG-1 isolates were also present. Between 2007 and 2008 canola leaves with characteristic blackleg lesions were collected from 54 fields located in the major canola growing regions of North Dakota. The virulence profile of 280 isolates retrieved from these leaves was characterized on the above mentioned set of differentials. The predominant species was L. maculans with approximately 74% of all isolates retrieved. Virulence profiles typical of PG-2, PG-3, PG-T, PG-4, and PG-5, which were previously described, were observed among the isolates evaluated. The virulence profile of almost 32% of all isolates did not fit within the previously described PGs and were assigned to new groups. The most prevalent PG among L. ma cu/ans isolates were PG-6 and PG-T. PG-6 causes a resistant reaction on differential 'Quinta'. PG-2, previously regarded as the most prevalent, was identified in only 13% of isolates. These results suggest a shift in the population of the blackleg species complex in North Dakota.
Studies on the Biology of Soybean Cyst Nematode
(North Dakota State University, 2011) Poromarto, Susilo Hambeg
Soybean cyst nematode (SCN), Heterodera glycines, is a threat to soybean production in North Dakota. Studies on the biology of SCN were conducted to improve my understanding and management of this plant parasitic nematode. The objectives of the research were to; (1) determine if SCN reproduces on crops commercially grown or being tested for production in North Dakota, (2) evaluate the effects of SCN on growth of dry bean, (3) determine if there could be a shift in the SCN population toward greater ability to reproduce on dry bean, and (4) characterize the spatial distribution of SCN in research size field experiments. Canola, clover, lentil, and sunflower were nonhosts while borage, camelina, chickpea, crambe, cuphea, field pea, nyjer, and safflower were poor hosts for SCN with female indices (FI) less than 8. Lupines were susceptible hosts with FI’s of 42 to 57. FI’s of dry bean cultivars varied from 5 to 117. Kidney beans averaged the highest FI at 110 followed by navy, pinto and black at FI’s 41, 39, and 16, respectively. Pod number (PN), pod weight (PW), seed number (SN), and seed weight (SW) of GTS-900 (pinto bean) were significantly less at 5,000 and 10,000 eggs/100 cm3 soil compared with the control by 44 to 56% averaged over the two years. Significant reduction in growth of Montcalm (kidney bean) and Mayflower (navy bean) was observed at 2,500 and 5,000 eggs/100 cm3 soils in 2009, but not in 2008. There was no evidence that SCN was increasing reproduction during two 11 month periods of continual reproduction on roots of dry bean cultivars Premiere and Cirrus (navy), Buster and Othello (pinto), and Eclipse and Jaguar (black). The spatial distribution of SCN in field plots was aggregated in nine of ten field sites with large differences in egg numbers between plots. Lloyd’s index of patchiness ranged from 1.09 to 3.34. Spatial distribution of SCN can be an important factor affecting the results of field experiments.
Detection of Qol Fungicide Resistance and Genomic Analysis for Identification of Polyketide Synthases and Methyltransferases in Ascochyta rabiei
(North Dakota State University, 2011) Delgado, Javier Andres
Ascochyta blight is the most important disease of chickpeas in North America since the fungal pathogen Ascochyta rabiei was introduced to this region in 1986. Ascochyta blight epidemics can cause yield and economic losses of up to 100%. Currently available chickpea cultivars are partially resistant to A. rabiei, and the disease is primarily managed with the aid of seed and foliar fungicide treatment throughout the growing season. Development of resistance to highly effective fungicides such as the quinone outside inhibitor (Qol) fungicides has therefore significantly threatened chickpea production in the US and Canada. In this dissertation, the mutation associated with resistance to Qol fungicides was identified in A. rabiei in North Dakota from which a quick and accurate diagnostic assay was developed for the detection of Qol resistant isolates of A. rabiei. The detection of Qol resistant isolates is important for designing management strategies aimed to controlling A. rabiei in the field. Polyketide synthases (PKSs) and methyltransferases have been associated with virulence in several fungi but these genes have not been identified or characterized previously in A. rabiei. Therefore, a bioinformatic resource was developed for the identification of PKS proteins from the genome of A. rabiei. This was developed using the 13-ketoacyl synthase and acyltransferase domains from PKS proteins exclusively belonging to the fungal species and was used to identify PKS genes from the A. rabiei genome. Several methyltransferase genes were also identified using a similar strategy. The characterization of the identified A. rabiei PKSs and methyltransferases was conducted to study the effect of the divalent cations present in chickpea seed extracts on the accumulation of phytotoxic compounds, vegetative growth, and conidial production. This study provides an insight into the effect of these divalent cations on the expression of genes that modulate biological processes that may be directly associated with infection and colonization of the host.
Genomics and Management of Fusarium Root Rot of Field Peas
(North Dakota State University, 2012) Chittem, Kishore
Dry Pea or field pea (Pisum sativum L.) is an important cool season legume crop grown in the United States. Field peas are vulnerable to many diseases of which, soil borne diseases including wilt and root rot are of major economic importance and can cause significant reduction in yield. There is a dearth of satisfactory methods for control of root rot and no varieties with complete resistance to Fusarium root rot are currently available. Root rot disease was found to be prevalent in all the major pea growing counties of North Dakota surveyed in 2004, 2005, 2010 and 2011. Fusarium species were the most frequently isolated fungal species from the infected pea roots of which, F. oxysporum and F. avenaceum were the most common. 21 Field pea varieties were screened for resistance against F. avenaceum and F. solani f. sp. pisi, the Fusarium species traditionally associated with root rots of field pea in growth chamber experiments and field trials. Low levels of resistance were detected in a few cultivars but no variety was found to be completely resistant to any of the pathogens tested. Efficiency of precipitated calcium carbonate (PCC) in controlling Fusarium species most commonly associated with root rots was evaluated under in vitro and field conditions. Significant reduction in spore production, spore germination, and dry mycelial weight of Fusarium spp. were detected on PCC amended media in laboratory studies. In greenhouse and field experiments significant reduction in root rot disease severity was observed with PCC application compared to control. Fungal gene expression in artificially infected field pea roots and F. graminearum grown in culture was assessed using the Illumina mRNA-Seq technology. A total of 613 F. graminearum genes were found to be differentially expressed in planta on pea. Functional classes associated with amino acid metabolism, nitrogen metabolism, extracellular polysaccharide degradation, detoxification by degradation and defense related proteins were found to be significantly enriched in the up-regulated gene set as determined using FunCatDB. Expression of four up-regulated genes was confirmed by RT-PCR to validate the inferences from the sequencing results.
Inoculation Techniques, Development of Brassica Napus Breeding Lines and Identification of Markers Associated with Resistance to Sclerotinia Sclerotiorum (Lib.) De Bary
(North Dakota State University, 2012) Burlakoti, Pragyan
Sclerotinia stem rot caused by Sclerotinia sclerotiorum (Lib.) de Bary is an economic disease affecting canola (Brassica napus L). Since expression of sclerotinia stem rot symptoms shows much variability and the trait is quantitative in nature, reliable phenotypic evaluation methods for characterization are needed. The three major objectives of this dissertation were to: i) evaluate eight different inoculation methods to discriminate between S. sclerotiorum-resistant and susceptible B. napus germplasm; ii) develop breeding lines with resistance to multiple diseases, and; iii) to identify QTL associated with resistance to sclerotinia stem rot using association mapping (AM). The eight methods evaluated were the detached leaves, detached stems, petiole inoculation (PIT), straw-inoculation, stem-piercing with toothpick, mycelial spray (MSI), petal inoculation and oxalic acid assay. MSI and PIT can better discriminate between the isolates and germplasm. Breeding lines resistance to S. sclerotiorum, Leptosphaeria maculans, and Rhizoctonia solani were developed from a cross between two moderately sclerotinia stem rot resistant plant introductions (PI). F2 seedlings were screened for sclerotinia stem rot using PIT. Surviving plants were self pollinated and their progeny screened again. This process was repeated until the F6 generation. In addition, F5 seedlings were evaluated for their reaction to R. solani and F5 and F6 seedlings for their reaction to L. maculans. Eight lines were identified as moderately resistance to these three pathogens. The genomes of a group of 278 B. napus plant introductions were screened using Diversity Array Technology to detect QTL associated with resistance to sclerotinia stem rot. The population was classified into nine sub-populations and 32 significant markers each explaining between 1.5 and 4.6% of the variation were identified. Blastn search indicates that similar nucleotide sequences are distributed throughout the genomes of B. oleracea, B. rapa, and A. thaliana. Results of these studies suggest the PIT and MSI are reliable screening tools to evaluate materials for resistance to sclerotinia stem rot; materials identified as resistant to S. sclerotiorum were also moderately resistant against R. solani and L. maculans and could be valuable sources for canola improvement programs; and AM allowed us to identify QTL associated with resistance to sclerotinia stem rot.
Characterization of Cytochrome B from European Field Isolates of Cercospora Beticola with Quinone Outside Inhibitor Resistance
(North Dakota State University, 2012) Birla, Keshav
Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is the most important foliar disease of sugar beet worldwide. Control strategies for CLS rely heavily on fungicides including quinone outside inhibitor (QOI) fungicides. We collected 866 C. beticola isolates from sugar beet growing regions in France and Italy and assessed their sensitivity to the QOI fungicide pyraclostrobin. To gain an understanding of the molecular basis of QOI resistance, we cloned the full-length coding region of Cbcytb. All tested QOI-resistant isolates harbored a point mutation in Cbcytb at nucleotide position 428 that conferred an exchange from glycine to alanine at amino acid position 143 (G143A). A PCR assay was developed to discriminate QOI-sensitive and QOI-resistant isolates based on the G143A mutation. Our results indicate that QOI resistance has developed in some European C. beticola populations in Italy and monitoring the G143A mutation is an essential fungicide resistance management strategy.
Movement and Accumulation of Candidatus Liberibacter Solanacearum in Potato Plants
(North Dakota State University, 2012) Rodriguez, Juan Jose
A new disease affecting potatoes was first detected in Mexico in 1993. Affected plants had aerial symptoms similar to those caused by potato purple top and psyllid yellows, but tubers had internal brown discoloration when sliced and dark stripes and streaks when processed to produce potato chips. The disease has been found in many potato production areas in Guatemala, Mexico, Honduras, New Zealand and the United States. The disease, termed Zebra Chip (ZC), has been associated with the presence of heavy infestations of the potato-tomato psyllid (Bactericera cockerelli). In 2009, a research group in New Zealand discovered that a new disease in tomato and pepper plants was caused by Candidatus Liberibacter solanacearum (Lso) and subsequently this same bacterium was associated with ZC in potato samples from Texas. The objectives of this study were: to assess the accumulation of Lso in various potato organs, to determine the effect of plant age on detection of Lso, symptom development and plant death, and (iii) to determine the effect of phosphorous acid on the development of ZC. Results from these studies showed significant differences in Lso populations between above and below ground tissues of the potato plant, with Lso populations in stolons and tubers being three to four times higher than those of leaf tissue and over seventy times greater than in stems. Time for detection of Lso by PCR in potato leaves of different ages at the time of inoculation ranged from 21 to 26 days after inoculation, symptoms development took 23 to 36 days. Plant death, took 24 to 47 days in plants of different age groups at the time of inoculation. In plants 15 weeks old at the time of inoculation, Lso was detected after 14 days in one plant out of 18; in plants 16 weeks old at the time of inoculation, Lso was detected after seven days in two plants out of 18. Phosphorous acid applications had no effect on the populations of Lso in potato tubers, onset of symptoms or plant death. All tubers showed ZC symptoms, making them unacceptable for the market.
Biology and Development of Two Wilt Fungi of Potato: Verticilliam Dahliae and Colletotrichum Coccodes
(North Dakota State University, 2012) Pasche, Julie Sherman
The demand for high quality tubers for the frozen processing industry has exerted increased pressure on producers to control diseases that can compromise tuber quality, including, C. coccodes and V. dahliae. Infection of potato plants by C. coccodes, the causal agent of black dot, can result in foliar necrosis, plant wilting, yield loss, tuber vascular discoloration and skin blemishes. Black dot of potato can originate from foliar-, seed- of soil-borne infections and, all plant tissues can be affected. The development of colonization in above- and below-ground plant tissues, from each inoculation/infestation source, was evaluated at two sites across two growing seasons. Colonization of potato tissue was detected at the first sampling date, even prior to plant emergence. While disease resulted from natural inoculum only, as inoculation/infestation sources increased, so did host colonization. Disease development also was greater in treatments with more than one site of inoculation/infestation. Overall, it was determined that stem tissue was colonized at greater frequency than root or stolon tissue. Infection of potato plants with V. dahliae, the causal agent of Verticillium wilt, occurs mainly through root contact with infested soil and can result in premature senescence as well as losses from decreased tuber yield and quality. Control of this pathogen is difficult and expensive and, therefore, efforts recently have increased towards the development of resistance. Resistance to V. dahliae in potato is thought to be multigenic, and therefore, quantification of the host:pathogen interaction is required to accurately define the level of resistance in a particular cultivar. Unfortunately, current methods used for quantification of V. dahliae in potato stems are time and labor intensive. A real-time duplex quantitative PCR assay was developed to simplify pathogen quantification to help breeding programs and researchers identify resistance in germplasm and cultivars. QPCR assays were validated using plant material from greenhouse and field trials, demonstrating specificity for V. dahliae, as well as sensitivity and accuracy when compared to traditional plating assays. Results from greenhouse and field evaluations also indicated that resistance is present among the eight russet-skinned cultivars evaluated in these studies.
Comparative Population Genetics of Fusarium Graminearum and Novel Sources of Resistance to Fusarium Head Blight in Spring Wheat
(North Dakota State University, 2013) Puri, Krishna Datta
Fusarium graminearum is the major causal agent of Fusarium head blight (FHB) in cereal crops. The fungus produces several types of trichothecenes [Deoxynivalenol (DON) and its acetylated derivatives, 3-acetyldeoxynivalenol (3ADON) and 15-acetyldeoxynivalenol (15ADON), and nivalenol (NIV)]. Characterization of 123 isolates collected during 1980 to 2000 (old collection), and 43 isolates collected in 2008 (new collection) from North Dakota revealed that 15ADON producing isolates were predominant (98%) in the old collection while the 3ADON producing isolates accounted for 43% in the new collection. Further, evaluation showed that the 3ADON isolates caused a higher disease severity and accumulated more DON in spring wheat than the 15ADON isolates. The 3ADON also exhibited higher DON in rice culture, and produced more spores on agar media. Population genetic analyses revealed a significant genetic differentiation between the two populations. To elucidate the transcriptomic differences between the two populations in vitro and in planta, RNA-sequencing was used. The in vitro gene expression comparison identified 479 up- and 801 down- regulated genes in the 3ADON population compared to 15ADON population. The in planta pair-wise comparisons between the two populations revealed 185, 89, and 62 unique genes to 3ADON at 48, 96 and 144 hours after inoculation (HAI), respectively. In a different study, population genetic analysis was conducted on 160 isolates collected in 2008 and 2009 from a FHB disease nursery located in China. All isolates analyzed were F. asiaticum except one (F. avenaceum). Of the 159 isolates, 79% were NIV producing, 18% were 15ADON and 3% were 3ADON. The two populations grouped based on year of collection exhibited low genetic differentiation (Fst = 0.032). To identify new sources of FHB resistance, 71 wheat accessions of diverse origins were re-evaluated for FHB severity and haplotyped using seventeen DNA markers associated with known resistance quantitative trait loci (QTL). Twenty two accessions had a haplotype different from all known sources used, suggesting that they may carry novel loci for FHB resistance. In conclusion, the information obtained in this study could have an impact on development of effective disease management measures and on improvement of FHB resistance in wheat.
Identification and Characterization of Pythium Spp. on Glycine Max (Soybean) in North Dakota
(North Dakota State University, 2013) Zitnick-Anderson, Kimberly Korthauer
The Oomycete Pythium comprises one of the most important groups of seedling pathogens affecting soybean, causing both pre- and post-emergence damping off. Numerous species of Pythium have been identified and found to be pathogenic on a wide range of hosts. Recent research on Pythium sp. infecting soybean has been limited to regions other than the Northern Great Plains and has not included North Dakota. In addition, little research has been conducted on the pathogenicity of various Pythium species on soybean or associations between Pythium communities and soil properties. Therefore, the objectives of this research were to isolate and identify the Pythium sp. infecting soybean in North Dakota, test their pathogenicity and assess if any associations between Pythium sp. and soil properties exist. Identification of the Pythium sp. was achieved using molecular techniques and morphological features. A total of 26 described Pythium sp. and several unknown species were recovered from soybean roots collected from 138 fields between 2011 and 2012. The majority of Pythium species (P. attrantheridium, P. debaryanum, P. diclinum, P. dissotocum, P. heterothallicum, P. hypogynum, P. inflatum, P. intermedium, P. irregulare, P. kashmirense, P. lutarium, P. minus, P. oopapillum, P. perplexum, P. terrestris, P. viniferum, P. violae,and an unknown Pythium sp.) caused pre-emergence damping off on soybean seedlings with less than 50% emergence and survival. In contrast, P. orthogonon, P. nunn, and P. rostratifingens had approximately 80% or greater emergence and survival of soybean seedlings. The negative and positive controls had 100% and 0% emergence and survival of soybean seedlings. Associations between soil properties and three Pythium groups were performed using logistic regression analysis. Logistic regression analysis determined that the presence of group one characterized by P. ultimum was correlated with zinc levels. Group two was characterized by P. kashmirense and an unknown Pythium sp. and was correlated with cation exchange capacity (CEC) values. Group three was characterized by P. irregulare and P. heterothallicum and was correlated with calcium carbonate exchange and CEC.
Developing Management Tools for Sunflower Rust (Puccinia Helianthi)
(North Dakota State University, 2013) Friskop, Andrew John
Sunflower rust, caused by Puccinia helianthi (Schwein), is an economically important disease in North Dakota. Since 2008, sunflower rust incidence and severity have increased. Therefore, a four year study was initiated to examine the management strategies for the disease. A total of 19 fungicide efficacy and timing trials were conducted from 2008-2011. Results indicated that DMI and QoI chemistries can be used effectively to manage the disease. Timing trials indicated that disease control was highest when fungicide applications were made at R5.0-R5.8. Results indicate a fungicide application is warranted when rust severities at approximately 1% are found on the upper-four leaves at R5. To observe phenotype variability in the pathogen, a P. helianthi survey was completed in 2011 and 2012. Single-pustule isolates were obtained and virulence phenotypes were evaluated on a set of nine differentials. Race characterization was assigned based on virulence phenotypes. In 2011, the most commonly detected races were 300 and 304, while the most virulent was 776. In 2012, races 304 and 324 were the most commonly detected and the most virulent was 777. To identify new sources of rust resistance, the core-set of Helianthus annuus germplasm was obtained from the USDA-North Central Regional Plant Introduction Station. The accession lines were screened both in the greenhouse and the field. The accessions were screened in the greenhouse individually to races 300, 304, 336, 337, and 777. Lines were screened in the field to a mixture of P. helianthi isolates coding to 300, 304, 336, and 337. The majority of lines were susceptible in both the field and greenhouse. At both field locations, PI 431538, PI 432512, and PI 650362 had year-end severities under 1%. Similarly, PI lines 432512 and 650362 had resistant infection types across all five races. The results of these studies provide information and tools that are being used currently to manage rust and will contribute to management in the future.
Genetic Structure of Leptosphaeria Maculans Populations in North Dakota and Identification of Genes Associated with Resistance to L. Maculans in Brassica Juncea
(North Dakota State University, 2013) Nepal, Achala
Blackleg, caused by Leptosphaeria maculans is one of the most devastating diseases of canola (Brassica napus) in North Dakota. A study was conducted to characterize prevalence of pathogenicity groups (PG), identify population structure of L. maculans and identify sources of resistance among B. juncea accessions. Approximately 56% of the isolates belonged to PG-4, 13% to PG-3, 11% to PG-T, 5% to PG-1, and 2% to PG2. The remaining 13% of isolates could not be identified. The 605 single-spore cultures used to study the population genetics of L. maculans in ND were grouped according to their county of origin in five regions (NE, NC, NW, WC, and C) and each region was considered a population. These populations were tested for genetic variation at 7 microsatellite, 4 minisatellite, and for mating type loci. High levels of genetic diversity (H = 0.63 to 0.70) and significant gametic disequilibrium (P < 0.001) was observed in all populations. The ratio of mating type idiomorphs deviated significantly (P < 0.05) from 1:1 ratio for four populations. Highly significant (P < 0.001) G''ST between pairs of populations indicated a strong population differentiation. STRUCTURE analysis identified three distinct genetic populations in which the majority of the isolates from WC and nearly half of the isolates from NC were assigned to subpopulation one. The remaining half of NC isolates were assigned to subpopulation two and rest were assigned to subpopulation three. To identify sources of resistance against PG-2, PG-3, PG-T, and PG-4, a set of 298 B. juncea accessions were screened in greenhouse trials. Six accessions were resistant to PG-2 and PG-3, nine accessions were resistant to PG-T and PG-4, and two accessions were resistant to PG-2 and PG-4. DNA extracted from these accessions was screened using 766 DArT markers to identify QTL associated with resistance. Thirteen DArT markers were significantly (P<0.05) associated with resistance to PG-2 with variability ranging from 0.9% to 6.4%. Of these, three were also found significantly (P<0.05) associated with resistance to PG-3 with variability of 4.4%. No markers were found to be significantly associated with resistance to PG-T or PG-4 at (P<0.05).
Identification of Multiple Virulence Loci in Pyrenophora Teres F. Teres Associated with Net Form Net Blotch in Barley
(North Dakota State University, 2013) Shjerve, Rachel Anne
The necrotrophic fungal pathogen Pyrenophora teres f. teres causes the foliar disease net form net blotch (NFNB) on barley (Hordeum vulgare). To investigate the genetics of virulence in the barley- P. teres f. teres pathosystem, we used 118 progeny derived from a cross between the isolates 15A and 6A. The barley lines, chosen based on their different reactions to 15A and 6A, were evaluated for NFNB caused by the 15A × 6A progeny. Genetic maps generated with SSR and AFLP markers in the fungal population were scanned for quantitative trait loci (QTL) associated with virulence in P. teres f. teres. Two QTL were identified in Rika, two in Kombar and PI356715 had a single unique QTL. Therefore, a total of five virulence loci were identified in this pathogen population based on inoculation on three different barley lines.
Understanding and Managing Rhizoctonia Solani in Sugarbeet
(North Dakota State University, 2013) Noor, Afsana
Rhizoctonia crown and root rot of sugarbeet (Beta vulgaris L.) caused by Rhizoctonia solani Kühn is one of the most important production problems in Minnesota and North Dakota. Greenhouse studies were conducted to determine the efficacy of azoxystrobin to control R. solani at seed, cotyledonary, 2-leaf and 4-leaf stages of sugarbeet; compatibility, safety, and efficacy of mixing azoxystrobin with starter fertilizers to control R. solani; and the effect of placement of azoxystrobin in control of R. solani. Results demonstrated that azoxystrobin provided effective control applied in-furrow or band applications before infection at all sugarbeet growth stages evaluated; mixtures of azoxystrobin and starter fertilizers were compatible, safe, and provided control of R. solani; and azoxystrobin provided effective control against R. solani when placed in contact over the sugarbeet root or into soil close to the roots.

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