Genomic regions associated with resistance to soybean rust (Phakopsora pachyrhizi) under field conditions in soybean germplasm accessions from Japan, Indonesia and Vietnam.

KEY MESSAGE: Eight soybean genomic regions, including six never before reported, were found to be associated with resistance to soybean rust (Phakopsora pachyrhizi) in the southeastern USA. Soybean rust caused by Phakopsora pachyrhizi is one of the most important foliar diseases of soybean [Glycine max (L.) Merr.]. Although seven Rpp resistance gene loci have been reported, extensive pathotype variation in and among fungal populations increases the importance of identifying additional genes and loci associated with rust resistance. One hundred and ninety-one soybean plant introductions from Japan, Indonesia and Vietnam, and 65 plant introductions from other countries were screened for resistance to P. pachyrhizi under field conditions in the southeastern USA between 2008 and 2015. The results indicated that 84, 69, and 49% of the accessions from southern Japan, Vietnam or central Indonesia, respectively, had negative BLUP values, indicating less disease than the panel mean. A genome-wide association analysis using SoySNP50K Infinium BeadChip data identified eight genomic regions on seven chromosomes associated with SBR resistance, including previously unreported regions of Chromosomes 1, 4, 6, 9, 13, and 15, in addition to the locations of the Rpp3 and Rpp6 loci. The six unreported genomic regions might contain novel Rpp loci. The identification of additional sources of rust resistance and associated genomic regions will further efforts to develop soybean cultivars with broad and durable resistance to soybean rust in the southern USA.

An Improved Crop Scouting Technique Incorporating Unmanned Aerial Vehicle-Assisted Multispectral Crop Imaging into Conventional Scouting Practice for Gummy Stem Blight in Watermelon.

Multispectral imaging is increasingly used in specialty crops, but its benefits in assessment of disease severity and improvements in conventional scouting practice are unknown. Multispectral imaging was conducted using an unmanned aerial vehicle (UAV), and data were analyzed for five flights from Florida and Georgia commercial watermelon fields in 2017. The fields were rated for disease incidence and severity by extension agents and plant pathologists at randomized locations (i.e., conventional scouting) followed by ratings at locations that were identified by differences in normalized difference vegetation index (NDVI) and stress index (i.e., UAV-assisted scouting). Diseases identified by the scouts included gummy stem blight, anthracnose, Fusarium wilt, Phytophthora fruit rot, Alternaria leaf spot, and cucurbit leaf crumple disease. Disease incidence and severity ratings were significantly different between conventional and UAV-assisted scouting (P < 0.01, Bhapkar/exact test). Higher severity ratings of 4 and 5 on a scale of 1 to 5 from no disease to complete loss of the canopy were more consistent after the scouts used the multispectral images in determining sampling locations. The UAV-assisted scouting locations had significantly lower green, red, and red edge NDVI values and higher stress index values than the conventional scouting areas (P < 0.05, ANOVA/Tukey), and this corresponded to areas with higher disease severity. Conventional scouting involving human evaluation remains necessary for disease validation. Multispectral imagery improved watermelon field scouting owing to increased ability to identify disease foci and areas of concern more rapidly than conventional scouting practices with early detection of diseases 20% more often using UAV-assisted scouting.

Relationship Between Piercing-Sucking Insect Control and Internal Lint and Seed Rot in Southeastern Cotton (Gossypium hirsutum L.).

In 1999, crop consultants scouting for stink bugs (Hemiptera spp.) in South Carolina discovered a formerly unobserved seed rot of cotton that caused yield losses ranging from 10 to 15% in certain fields. The disease has subsequently been reported in fields throughout the southeastern Cotton Belt. Externally, diseased bolls appeared undamaged; internally, green fruit contain pink to dark brown, damp, deformed lint, and necrotic seeds. In greenhouse experiments, we demonstrated transmission of the opportunistic bacterium Pantoea agglomerans by the southern green stink bug, Nezara viridula (L.). Here, green bolls were sampled from stink bug management plots (insecticide protected or nontreated) from four South Atlantic coast states (North Carolina, South Carolina, Georgia, and Florida) to determine disease incidence in the field and its association with piercing-sucking insects feeding. A logistic regression analysis of the boll damage data revealed that disease was 24 times more likely to occur (P = 0.004) in bolls collected from plots in Florida, where evidence of pest pressure was highest, than in bolls harvested in NC with the lowest detected insect pressure. Fruit from plots treated with insecticide, a treatment which reduced transmission agent numbers, were 4 times less likely to be diseased than bolls from unprotected sites (P = 0.002). Overall, punctured bolls were 125 times more likely to also have disease symptoms than nonpunctured bolls, irrespective of whether or not plots were protected with insecticides (P = 0.0001). Much of the damage to cotton bolls that is commonly attributed to stink bug feeding is likely the resulting effect of vectored pathogens.

From Select Agent to an Established Pathogen: The Response to Phakopsora pachyrhizi (Soybean Rust) in North America.

The pathogen causing soybean rust, Phakopsora pachyrhizi, was first described in Japan in 1902. The disease was important in the Eastern Hemisphere for many decades before the fungus was reported in Hawaii in 1994, which was followed by reports from countries in Africa and South America. In 2004, P. pachyrhizi was confirmed in Louisiana, making it the first report in the continental United States. Based on yield losses from countries in Asia, Africa, and South America, it was clear that this pathogen could have a major economic impact on the yield of 30 million ha of soybean in the United States. The response by agencies within the United States Department of Agriculture, industry, soybean check-off boards, and universities was immediate and complex. The impacts of some of these activities are detailed in this review. The net result has been that the once dreaded disease, which caused substantial losses in other parts of the world, is now better understood and effectively managed in the United States. The disease continues to be monitored yearly for changes in spatial and temporal distribution so that soybean growers can continue to benefit by knowing where soybean rust is occurring during the growing season.

Effect of solar radiation on severity of soybean rust.

Soybean rust (SBR), caused by Phakopsora pachyrhizi, is a damaging fungal disease of soybean (Glycine max). Although solar radiation can reduce SBR urediniospore survival, limited information is available on how solar radiation affects SBR progress within soybean canopies. Such information can aid in developing accurate SBR prediction models. To manipulate light penetration into soybean canopies, structures of shade cloth attenuating 30, 40, and 60% sunlight were constructed over soybean plots. In each plot, weekly evaluations of severity in lower, middle, and upper canopies, and daily temperature and relative humidity were recorded. Final plant height and leaf area index were also recorded for each plot. The correlation between amount of epicuticular wax and susceptibility of leaves in the lower, middle, and upper canopies was assessed with a detached leaf assay. Final disease severity was 46 to 150% greater in the lower canopy of all plots and in the middle canopy of 40 and 60% shaded plots. While daytime temperature within the canopy of nonshaded soybean was greater than shaded soybean by 2 to 3°C, temperatures recorded throughout typical evenings and mornings of the growing season in all treatments were within the range (10 to 28.5°C) for SBR development as was relative humidity. This indicates temperature and relative humidity were not limiting factors in this experiment. Epicuticular wax and disease severity in detached leaf assays from the upper canopy had significant negative correlation (P = 0.009, R = -0.84) regardless of shade treatment. In laboratory experiments, increasing simulated total solar radiation (UVA, UVB, and PAR) from 0.15 to 11.66 MJ m(-2) increased mortality of urediniospores from 2 to 91%. Variability in disease development across canopy heights in early planted soybean may be attributed to the effects of solar radiation not only on urediniospore viability, but also on plant height, leaf area index, and epicuticular wax, which influence disease development of SBR. These results provide an understanding of the effect solar radiation has on the progression of SBR within the soybean canopy.

Evolution of Bacillus subtilis to enhanced growth at low pressure: up-regulated transcription of des-desKR, encoding the fatty acid desaturase system.

The atmospheric pressure on Mars ranges from 1-10 mbar, about 1% of Earth pressure (∼1013 mbar). Low pressure is a growth-inhibitory factor for terrestrial microorganisms on Mars, and a putative low-pressure barrier for growth of Earth bacteria of ∼25 mbar has been postulated. In a previous communication, we described the isolation of a strain of Bacillus subtilis that had evolved enhanced growth ability at the near-inhibitory low pressure of 50 mbar. To explore mechanisms that enabled growth of the low-pressure-adapted strain, numerous genes differentially transcribed between the ancestor strain WN624 and low-pressure-evolved strain WN1106 at 50 mbar were identified by microarray analysis. Among these was a cluster of three candidate genes (des, desK, and desR), whose mRNA levels in WN1106 were higher than the ancestor on the microarrays. Up-regulation of these genes was confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The des, desK, and desR genes encode the Des membrane fatty acid (FA) desaturase, the DesK sensor kinase, and the DesR response regulator, respectively, which function to maintain membrane fluidity in acute response to temperature downshift. Pressure downshift caused an up-regulation of des mRNA levels only in WN1106, but expression of a des-lacZ transcriptional fusion was unaffected, which suggests that des regulation was different in response to temperature versus pressure downshift. Competition experiments showed that inactivation of the des gene caused a slight, but statistically significant, loss of fitness of strain WN1106 at 50 mbar. Further, analysis of membrane FA composition of cells grown at 1013 versus 50 mbar revealed a decrease in the ratio of unsaturated to saturated FAs but an increase in the ratio of anteiso- to iso-FAs. The present study represents a first step toward identification of molecular mechanisms by which B. subtilis could sense and respond to the novel environmental stress of low pressure.

Epidemiology of Soybean Rust in Soybean Sentinel Plots in Florida.

Since its discovery in the southeastern United States in 2004, soybean rust (SBR) has been variable from year to year. Caused by Phakopsora pachyrhizi, SBR epidemics in Florida are important to understand, as they may serve as an inoculum source for other areas of the country. This study examined the first disease detection date, incidence, and severity of SBR in relation to environmental data, growth stage, and maturity group (MG3, MG5, MG7) in soybean sentinel plots (225 m2) across north Florida from 2005 through 2008. The majority (91%) of the initial infections were observed in MG5 and MG7 soybeans, with plots not becoming infected until growth stage R4 or later. Precipitation was the principle factor affecting disease progress, where disease increased rapidly after rain events and was suppressed during dry periods. On average, plots became infected 30 days earlier in 2008 than 2005. In 2008, there was a significant increase in disease incidence and severity associated with the occurrence of Tropical Storm Fay, which deposited up to 380 mm of rainfall in north Florida. The results of this study indicate that climatic and environmental factors are important in determining the development of SBR in north Florida.

Combinatorially selected peptides for protection of soybean against Phakopsora pachyrhizi.

Phakopsora pachyrhizi, the fungal pathogen that causes Asian soybean rust, has the potential to cause significant losses in soybean yield in many production regions of the United States. Germplasm with durable, single-gene resistance is lacking, and control of rust depends on timely application of fungicides. To assist the development of new modes of soybean resistance, we identified peptides from combinatorial phage-display peptide libraries that inhibit germ tube growth from urediniospores of P. pachyrhizi. Two peptides, Sp2 and Sp39, were identified that inhibit germ tube development when displayed as fusions with the coat protein of M13 phage or as fusions with maize cytokinin oxidase/dehydrogenase (ZmCKX1). In either display format, the inhibitory effect of the peptides on germ tube growth was concentration dependent. In addition, when peptides Sp2 or Sp39 in either format were mixed with urediniospores and inoculated to soybean leaves with an 8-h wetness period, rust lesion development was reduced. Peptides Sp2 and Sp39, displayed on ZmCKX1, were found to interact with a 20-kDa protein derived from germinated urediniospores. Incorporating peptides that inhibit pathogen development and pathogenesis into breeding programs may contribute to the development of soybean cultivars with improved, durable rust tolerance.

Effects of Surface Wetness Periods on Development of Soybean Rust Under Field Conditions.

Soybean rust (SBR), caused by Phakopsora pachyrhizi, has the potential to be an economic threat to U.S. soybean production after its arrival to the continental United States in 2004. The use of fungicides to control SBR may be problematic due to the large acreage that needs to be protected, the high costs of fungicides, and the cost of application. Cultural practices such as the use of reduced seed rates, increased row widths, and row orientation to the sun have been prescribed as environmental modifications that create a microclimate less conducive to foliar disease development. Therefore, our objective was to determine the influence of different periods of leaf wetness and respective microenvironments on infection and rust development on soybean plants in the field. A misting irrigation system was developed and applied on MGV soybean for 1 min every 30 min for 0-, 6-, 12-, and 18-h periods. This study indicates that extended periods of leaf wetness (18 h) increase disease severity and the rate of spread of the disease in the upper canopy. These results, in combination with spore monitoring, may be used to refine models of pathogen reproduction, prediction, and risk in a certain regions.

Population dynamics and within-plant distribution of Frankliniella spp. thrips (Thysanoptera: Thripidae) in cotton.

Thrips cause damage to seedling cotton, and they are associated with the spread of Fusarium hardlock, a serious disease affecting cotton bolls that reduces lint yield in the southern United States. The population dynamics and within-plant distribution of Frankliniella spp. thrips in cotton were determined in 2005, 2006, and 2007 in Quincy and in 2006 and 2007 in Marianna, FL. Frankliniella tritici (Fitch) was >98% of the adult thrips collected. The adults of F. bispinosa (Morgan), F. occidentalis (Pergande), and F. fusca (Hinds) also were collected. Populations of F. fusca inhabited the cotton leaves of vegetative-stage cotton. The other species were highly anthophilic, primarily inhabiting cotton during its reproductive growth stages. Densities of these species peaked during mid-season, which also coincided with peak bloom. The adult F. tritici and the thrips larvae were aggregated in the flowers over the other plant parts, with more collected in the upper than the lower canopies. These patterns of aggregation were observed at each location each year, and the numerical differences usually were significant (P < 0.05). Densities of adult F. tritici were low on the leaves, squares, and bolls. The population dynamics and with-plant distribution of individual thrips species has not previously been determined for cotton in the southern United States.

Flower-inhabiting Frankliniella Thrips (Thysanoptera: Thripidae), pesticides, and Fusarium hardlock in cotton.

Cotton hardlock caused by Fusarium verticillioides (Sacc. Nirenberg) can reduce cotton, Gossypium hirsutum L., yields > 70% in the southeastern United States. The spores infect flowers on the day of pollination, resulting in hardlock, which is the failure of the fiber to fluff as the boll opens at maturity. Frankliniella spp. Thrips (Thysanoptera: Thripidae) inhabiting the flowers are hypothesized to increase hardlock by spreading the conidia or by creating entranceways for the germinating Fusarium conidia. Experiments were conducted at Marianna and Quincy in Florida in 2006 and 2007 to determine whether there was a relationship between the number of adult and larval thrips inhabiting the flowers of cotton and the incidence of cotton hardlock. Frankliniella tritici (Fitch) was > 98% of the adult thrips in the samples at both locations each year. The adults of Frankliniella bispinosa (Morgan) and Frankliniella occidentalis (Pergande) also were collected. There were no significant regression relationships between weekly mean densities of thrips in the flowers and the incidence of cotton hardlock at harvest in any of the experiments. Additional experiments were conducted at each location in 2006 and 2007 to determine whether weekly applications during flowering of the insecticide lambda-cyhalothrin, the fungicide thiophanate methyl, and the combination of the two reduced the incidence of cotton hardlock at harvest. Applications of the insecticide significantly reduced the numbers of adult F. tritici, the number of thrips larvae, and the incidence of hardlock at harvest. Applications of the insecticide were as affective as applications of the insecticide plus fungicide. In one experiment, applications of the fungicide reduced the incidence of hardlock at harvest. Applications of the insecticide usually significantly increased the number of adult F. occidentalis. None of the pesticide treatments significantly affected the numbers of the key thrips predator Orius insidiosus (Say). We conclude that insecticidal control of the adults and larvae of F. tritici during flowering reduced the incidence of cotton hardlock. However, there were no significant regression relationships between the incidence of cotton hardlock at harvest and the number of thrips in the flowers.

Differential expression of genes in soybean in response to the causal agent of Asian soybean rust (Phakopsora pachyrhizi Sydow) is soybean growth stage-specific.

Understanding plant host response to a pathogen such as Phakopsora pachyrhizi, the causal agent of Asian soybean rust (ASR), under different environmental conditions and growth stages is crucial for developing a resistant plant variety. The main objective of this study was to perform global transcriptome profiling of P. pachyrhizi-exposed soybean (Glycine max) with susceptible reaction to the pathogen from two distinct developmental growth stages using whole genome Affymetrix microarrays of soybean followed by confirmation using a resistant genotype. Soybean cv. 5601T (susceptible to ASR) at the V(4) and R(1) growth stages and Glycine tomentella (resistant to ASR) plants were inoculated with P. pachyrhizi and leaf samples were collected after 72 h of inoculation for microarray analysis. Upon analyzing the data using Array Assist software at 5% false discovery rate (FDR), a total of 5,056 genes were found significantly differentially expressed at V(4) growth stage, of which 2,401 were up-regulated, whereas 579 were found differentially expressed at R(1) growth stage, of which 264 were up-regulated. There were 333 differentially expressed common genes between the V(4) and R(1) growth stages, of which 125 were up-regulated. A large difference in number of differentially expressed genes between the two growth stages indicates that the gene expression is growth-stage-specific. We performed real-time RT-PCR analysis on nine of these genes from both growth stages and both plant species and found results to be congruent with those from the microarray analysis.

Winter Survival of the Soybean Rust Pathogen, Phakopsora pachyrhizi, in Florida.

Soybean rust (SBR) survival and host availability (kudzu, Pueraria spp.) were assessed from November 2006 through April 2007 at six sites from the panhandle to southwest Florida. Micro loggers recorded both temperature and relative humidity hourly at each location. Periods of drought and cumulative hours below 0°C correlated with kudzu defoliation. Inoculum potential from detached kudzu leaves was evaluated in vitro under various temperature and relative humidity levels. Kudzu leaves with SBR kept at 4°C produced viable urediniospores with the highest germination at all moisture levels over time. Freezing temperatures (-4 and -20°C) drastically reduced spore germination. However, when leaves were incubated at low (<35%) relative humidity, inoculum potential was prolonged. Results from this study demonstrate that both temperature and relative humidity impact P. pachyrhizi in the field and in vitro, and that detached kudzu leaves have the potential to serve as an inoculum source in kudzu stands.