Effects of Rag1 aphid‐resistant soybean on mortality,development, and preference of brown marmorated stink bug

The brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), poses a new threat to soybean, Glycine max (L.) Merrill (Fabaceae), production in the north central USA. As H. halys continues to spread and increase in abundance in the region, the interaction between H. halys and management tactics deployed for other pests must be determined. Currently, the soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is the most abundant and damaging insect pest of soybean in the region. Aphid‐resistant soybean, mainly with the Rag1 gene, is commercially available for management of A. glycines. Here, experiments were performed to evaluate the effects of Rag1 aphid‐resistant soybean on the mortality, development, and preference of H. halys. In a no‐choice test, mortality of H. halys reared on Rag1 aphid‐resistant soybean pods was significantly lower than when reared on aphid‐susceptible soybean pods (28 vs. 53%). Development time, adult weight, and proportion females of surviving adults did not differ when reared on Rag1 aphid‐resistant or aphid‐susceptible soybean pods. In choice tests, H. halys exhibited a preference for Rag1 aphid‐resistant over aphid‐susceptible soybean pods after 4 h, but not after 24 h. Halyomorpha halys exhibited no preference when tested with vegetative‐stage or reproductive‐stage soybean plants. The preference by H. halys for Rag1 aphid‐resistant soybean pods and the decreased mortality when reared on these pods suggests that the use of Rag1 aphid‐resistant soybean may favor this emerging pest in the north central USA.     

Genetic parameters and selection strategies for soybean genotypes resistant to the stink bug-complex

Soybean genotypes resistant to stink bugs are derived from complex breeding processes obtained through indirect selection. The aim of the present work was to estimate genetic parameters for guiding selection strategies towards resistant genotypes, based on those traits associated with responses to pod-attacking stink bugs, such as the grain filling period (GFP), leaf retention (LR), percentage index of pod damage (PIPD) and percentage of spotted seeds (PSS). We assessed the parental lines IAC-100 (resistant) and FT-Estrela (susceptible), the progenies F(2) and F (4) , 30 progenies F (2:3) , 30 progenies BC (1) F (2:3) and 30 progenies BC (2) F (2:3) , besides the cultivars BRS Celeste and MGBR-46 (Conquista). Three field experiments, using randomized complete block design with three replications, were installed in Goiania-GO, in the 2002/03 season. Each experiment consisted of 36 treatments (6 common and 30 regular). Heritability estimates were: 74.6 and 36.1 (GFP); 51.9 and 19.9 (LR); 49.6 and 49.6 (PIPD) and 55.8 and 20.3 (PSS), in both the broad and narrow senses, respectively. Based on these results, we concluded that the best strategy for obtaining stink bug-resistant genotypes consists of selecting the PIPD trait in early generations (F (3) or F (4) ), followed by selection for the GFP, LR and PSS traits in generations with higher endogamy levels.     

Waveform characterization of the soybean stem feeder Edessa meditabunda: overcoming the challenge of wiring pentatomids for EPG

Stink bugs (Hemiptera: Heteroptera: Pentatomidae) are in general robust and restless insects, which makes them difficult to wire for electropenetrograph (EPG) studies. In addition, cuticular lipids may reduce wire effectiveness, and their removal could improve success of wiring. We compared wiring effectiveness for three species of stink bugs, differing in walking behaviour and degree of cuticular waxiness, that is, Piezodorus guildinii (Westwood), Nezara viridula (L.), and Loxa deducta (Walker). Results indicated that removal of cuticular lipids by mechanical abrasion (via sanding) greatly improved attachment success with gold wire. Our hypothesis that heavier and bigger bugs would lose the wire attachment more quickly than lighter and smaller bugs was not confirmed, regardless of the sanding. In contrast, our hypothesis that greater movement of a bug would cause the wire to break more often was supported by extensive testing. Behaviour appears to be more relevant for successful wiring than body weight. We used the sanding and wiring technique to characterize and correlate direct current EPG waveforms for the large and restless stem‐feeding stink bug Edessa meditabunda (Fabricius) on soybean plants. This marks the first published example of pentatomid EPG waveforms. Edessa meditabunda recordings on soybean stems generated eight types of waveforms in three phases and two families, named as follows: non‐probing = Np and Z; pathway phase = Em1; X wave phase = X; ingestion phase, family I = Em2 and Em3; ingestion phase, family N = Em4 and Em5. These eight were described based on their frequencies, relative amplitudes, and level voltages. Histological studies of stylets within salivary sheaths correlated the Em1, Em2, and Em3 waveforms with specific penetration sites. The waveform with the longest duration when feeding was Em2, representing xylem sap ingestion; in addition, waveform Em3 (always preceded by an X wave) was correlated with phloem sap ingestion.     

Construction and comparison of three reference‐quality genome assemblies for soybean

We report reference‐quality genome assemblies and annotations for two accessions of soybean (Glycine max) and for one accession of Glycine soja, the closest wild relative of G. max. The G. max assemblies provided are for widely used US cultivars: the northern line Williams 82 (Wm82) and the southern line Lee. The Wm82 assembly improves the prior published assembly, and the Lee and G. soja assemblies are new for these accessions. Comparisons among the three accessions show generally high structural conservation, but nucleotide difference of 1.7 single‐nucleotide polymorphisms (snps) per kb between Wm82 and Lee, and 4.7 snps per kb between these lines and G. soja. snp distributions and comparisons with genotypes of the Lee and Wm82 parents highlight patterns of introgression and haplotype structure. Comparisons against the US germplasm collection show placement of the sequenced accessions relative to global soybean diversity. Analysis of a pan‐gene collection shows generally high conservation, with variation occurring primarily in genomically clustered gene families. We found approximately 40–42 inversions per chromosome between either Lee or Wm82v4 and G. soja, and approximately 32 inversions per chromosome between Wm82 and Lee. We also investigated five domestication loci. For each locus, we found two different alleles with functional differences between G. soja and the two domesticated accessions. The genome assemblies for multiple cultivated accessions and for the closest wild ancestor of soybean provides a valuable set of resources for identifying causal variants that underlie traits for the domestication and improvement of soybean, serving as a basis for future research and crop improvement efforts for this important crop species.     

Stacking of a stearoyl‐ACP thioesterase with a dual‐silenced palmitoyl‐ACP thioesterase and ∆12 fatty acid desaturase in transgenic soybean

Soybean (Glycine max (L.) Merr) is valued for both its protein and oil, whose seed is composed of 40% and 20% of each component, respectively. Given its high percentage of polyunsaturated fatty acids, linoleic acid and linolenic acid, soybean oil oxidative stability is relatively poor. Historically food processors have employed a partial hydrogenation process to soybean oil as a means to improve both the oxidative stability and functionality in end‐use applications. However, the hydrogenation process leads to the formation of trans‐fats, which are associated with negative cardiovascular health. As a means to circumvent the need for the hydrogenation process, genetic approaches are being pursued to improve oil quality in oilseeds. In this regard, we report here on the introduction of the mangosteen (Garcinia mangostana) stearoyl‐ACP thioesterase into soybean and the subsequent stacking with an event that is dual‐silenced in palmitoyl‐ACP thioesterase and ?12 fatty acid desaturase expression in a seed‐specific fashion. Phenotypic analyses on transgenic soybean expressing the mangosteen stearoyl‐ACP thioesterase revealed increases in seed stearic acid levels up to 17%. The subsequent stacked with a soybean event silenced in both palmitoyl‐ACP thioesterase and ?12 fatty acid desaturase activity, resulted in a seed lipid phenotype of approximately 11%–19% stearate and approximately 70% oleate. The oil profile created by the stack was maintained for four generations under greenhouse conditions and a fifth generation under a field environment. However, in generation six and seven under field conditions, the oleate levels decreased to 30%–40%, while the stearic level remained elevated.     

Global investigation of the co‐evolution of MIRNA genes and microRNA targets during soybean domestication

Although the selection of coding genes during plant domestication has been well studied, the evolution of MIRNA genes (MIRs) and the interaction between microRNAs (miRNAs) and their targets in this process are poorly understood. Here, we present a genome‐wide survey of the selection of MIRs and miRNA targets during soybean domestication and improvement. Our results suggest that, overall, MIRs have higher evolutionary rates than miRNA targets. Nonetheless, they do demonstrate certain similar evolutionary patterns during soybean domestication: MIRs and miRNA targets with high expression and duplication status, and with greater numbers of partners, exhibit lower nucleotide divergence than their counterparts without these characteristics, suggesting that expression level, duplication status, and miRNA–target interaction are essential for evolution of MIRs and miRNA targets. Further investigation revealed that miRNA–target pairs that are subjected to strong purifying selection have greater similarities than those that exhibited genetic diversity. Moreover, mediated by domestication and improvement, the similarities of a large number of miRNA–target pairs in cultivated soybean populations were increased compared to those in wild soybeans, whereas a small number of miRNA–target pairs exhibited decreased similarity, which may be associated with the adoption of particular domestication traits. Taken together, our results shed light on the co‐evolution of MIRs and miRNA targets during soybean domestication.     

Seasonal abundance of the neotropical brown stink bug, Euschistus heros, in overwintering sites, and the breaking of dormancy

The seasonal abundance of the neotropical brown stink bug, Euschistus heros (Heteroptera: Pentatomidae), in overwintering sites in northern Paraná state, Brazil (latitude 23°11 S, longitude 51°11 W) was monitored from September 1994 to August 1995. The breaking of dormancy (oligopause) was studied in the laboratory by comparing the feeding activity and reproduction of adults collected in the field under different physiological conditions (i.e., dormant and non-dormant). No bugs were found in overwintering sites during the summer (December to February) and during early autumn (March). From mid-autumn to winter (April – August), the number of E. heros captured in these sites gradually increased, decreasing thereafter with the start of spring in September. Dormant and non-dormant E. heros taken to the laboratory and maintained at 25 ± 1 °C , 65± 5% r.h., and L14: D10 photoperiod, and given suitable food (soybean pods or seeds), began feeding immediately. The number of stylet sheaths deposited/day on the food was greater for non-dormant than for dormant adults. Feeding activity was greater on immature pods than on mature seeds of soybean. Dormant females placed under suitable biotic and abiotic conditions took ca. 2 weeks to start reproduction, in contrast to non-dormant females, which reproduced immediately.     

Insecticidal potential of Bacillus thuringiensis for the biological control of neotropical brown stink bug

Euschistus heros (Hemiptera: Pentatomidae) is considered a major insect pest in soybean production in Brazil. The indiscriminate application of pesticides to fields leads to reduction in biodiversity, selection of resistant populations, emergence of new pest outbreaks, and damage to non‐target organisms. The objective of this research was to evaluate the effects of exposure to Cry proteins present in new isolates of Bacillus thuringiensis Berliner, commercial formulations, and Bt soybean on E. heros development. The feeding preference of E. heros for Bt‐ vs. non‐Bt soybean was also evaluated. All treatments caused significant mortality to E. heros except the Bt soybean. Tests with combinations of isolated Bt‐toxins indicated that combinations had greater efficacy than other treatments (>98% mortality). The results demonstrate that E. heros is susceptible to B. thuringiensis toxins, which may contribute to the management of this insect in soybean agro‐ecosystems.     

Distribution of Bemisia tabaci within soybean plants and on individual leaflets

To control whiteflies on soybean crops in an effective and economically viable way, it is necessary to quantify the occurrence and density of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) on the leaflets. Estimating the number of B. tabaci cm^‐2 on leaflets is difficult, because its distribution pattern on the various parts of the plant canopy and on the leaflet surface is unknown. The aim of this study was to evaluate the distribution of B. tabaci nymphs on soybean plants and leaflets, under greenhouse and field conditions. One hundred soybean plants infested with all nymph stages were randomly selected in a greenhouse, and 25 in a field. Of each plant, a trifoliate leaf of the middle third of the plant’s height was selected and its central leaflet was collected (greenhouse experiment), or a trifoliate leaf of each third layer (upper, middle, and lower), of which the left, central, and right leaflets were collected (field experiment). The collected leaflets were divided into 32 sections (1 cm² per section), arranged in an array of eight rows and four columns to count whitefly nymphs. The Morisita index (I_δ), the negative binomial parameter k, and the dispersion index (I) were calculated for each leaflet, using the number of nymphs as variable. The highest population densities of whitefly nymphs were found in the middle third of the soybean plants. In leaflets from the middle third, the nymphs concentrated in the middle and bottom parts of the leaflets, whereas in the upper and lower thirds of the plant, they were randomly distributed on the leaflets.     

Leaf gas exchange and chlorophyll a fluorescence in soybean leaves infected by Phakopsora pachyrhizi

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is one of the most important foliar diseases affecting soybean production worldwide. This study aimed to investigate the photosynthetic performance (leaf gas exchange, chlorophyll (Chl) a fluorescence images and photosynthetic pigment pools) of soybean plants sprayed with Acibenzolar‐S‐Methyl (ASM) and the fungicide epoxiconazole + pyraclostrobin (Epo+Pyr) and further inoculated with P. pachyrhizi. The ASR symptoms progressed much faster on the leaves of plants from the control treatment (water spray) in comparison with the ASM and Epo+Pyr treatments. In general, the values for the leaf gas exchange parameters net carbon assimilation rate (A), stomatal conductance to water vapour (g_s), internal CO₂ concentration (C_i) and transpiration rate (E) increased for the infected plants sprayed with ASM or Epo+Pyr in comparison with plants from the control treatment. The values for the initial fluorescence (F_o), maximal fluorescence (F_m), maximal photosystem II quantum efficiency (F_v/F_m), effective photosystem II quantum yield (Y(II)) and quantum yield of regulated energy dissipation (Y(NPQ)) were consistently higher for the ASM and Epo+Pyr treatments in comparison with the control treatment at advanced stages of fungal infection. By contrast, the values for quantum yield of non‐regulated energy dissipation (Y(NO) were significantly lower for the ASM and Epo+Pyr treatments. The concentrations of total Chl a+b and carotenoids significantly increased for infected plants sprayed with ASM and Epo+Pyr in comparison with plants from the control treatment. The results of this study demonstrated that the spray of soybean plants with either ASM or Epo+Pyr contributed to reduce the negative effect of ASR on the photosynthesis of soybean plants.     

Anatomical changes in stem and root of soybean plants submitted to salt stress

• The soybean is a legume that is widely cultivated in many countries due to the high levels of protein and oil contained in its seed, and is used for human and animal nutrition. However, salinity affects more than 800 million hectares worldwide, limiting global agricultural production.
• The aim of this research was to evaluate the structural behaviour of the roots and stems under progressive salt stress, detailing the possible anatomical modifications to these organs in soybean plants during this stress. The plants were randomized into five treatments (0, 50, 100, 150 and 200 mm NaCl).
• All the root regions studied and exposed to 100 mm Na⁺ exhibited increases in the epidermis and endodermis and formation of lysogenic aerenchyma with increasing salinity, revealing the protective roles of these structures in reducing Na⁺ influx. In the stem, increases in the cortex and pith in the first internode subject to 100 mm Na⁺ suggest anatomical responses that aim to minimize oxidative stress.
• Soybean plants subjected to progressive salt stress (>50 mm Na⁺) avoided cavitation and loss of function linked to vessel elements, reducing the metaxylem in all the root and stem regions analysed. Finally, our results confirm anatomical changes to the roots and stems.

     

Roles of small RNAs in soybean defense against Phytophthora sojae infection

The genus Phytophthora consists of many notorious pathogens of crops and forestry trees. At present, battling Phytophthora diseases is challenging due to a lack of understanding of their pathogenesis. We investigated the role of small RNAs in regulating soybean defense in response to infection by Phytophthora sojae, the second most destructive pathogen of soybean. Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are universal regulators that repress target gene expression in eukaryotes. We identified known and novel small RNAs that differentially accumulated during P. sojae infection in soybean roots. Among them, miR393 and miR166 were induced by heat‐inactivated P. sojae hyphae, indicating that they may be involved in soybean basal defense. Indeed, knocking down the level of mature miR393 led to enhanced susceptibility of soybean to P. sojae; furthermore, the expression of isoflavonoid biosynthetic genes was drastically reduced in miR393 knockdown roots. These data suggest that miR393 promotes soybean defense against P. sojae. In addition to miRNAs, P. sojae infection also resulted in increased accumulation of phased siRNAs (phasiRNAs) that are predominantly generated from canonical resistance genes encoding nucleotide binding‐leucine rich repeat proteins and genes encoding pentatricopeptide repeat‐containing proteins. This work identifies specific miRNAs and phasiRNAs that regulate defense‐associated genes in soybean during Phytophthora infection.     

Standardized sampling plan for Aphis gossypii based on the cotton cultivar,plant phenology and crop size

A standardized sampling plan is the starting point for developing a decision‐making system for pest control. Aphis gossypii (Hemiptera: Aphididae) is a destructive sap‐feeding pest on cotton worldwide. However, research addressing cotton cultivar, plant phenology and field size with the aim of developing a sampling plan for A. gossypii has not been done. Therefore, in this study, we developed a standardized sampling for A. gossypii as a function of these factors. To accomplish this, A. gossypii densities in four experimental cotton cultivars were sampled weekly during year one to determine the ideal aphid characteristic to sample (by individual or colony). During year one and two, A. gossypii densities were sampled weekly in the same cultivars to determine sampling unit, sampling technique and the number of samples for an A. gossypii sampling plan. Using the sample number determined, the sampling time was recorded for cotton field size of 1, 5, 10, 50, 100 and 150 ha in order to estimate the sampling cost. In cotton, the count of individuals was the best characteristic for the assessment of A. gossypii. Leaves of the most apical branches for the vegetative and reproductive cotton plant stage were the best sampling units. The best sampling technique was direct counting. The cotton cultivar did not affect the development of the sampling plan. The A. gossypii sampling plan involved the evaluation of 58 samples per zone and required 20 min (<0.35 min/sample) for the evaluation of these samples. However, the walking time between samples was the main factor responsible for the total sampling time and cost in cotton fields, and this factor strongly depends on the size of the cotton field.     

Osmotic stress decreases complexity underlying the electrophysiological dynamic in soybean

• Studies on plant electrophysiology are mostly focused on specific traits of single cells. Inspired by the complexity of the signalling network in plants, and by analogy with neurons in human brains, we sought evidence of high complexity in the electrical dynamics of plant signalling and a likely relationship with environmental cues.
• An EEG‐like standard protocol was adopted for high‐resolution measurements of the electrical signal in Glycine max seedlings. The signals were continuously recorded in the same plants before and after osmotic stimuli with a ?2 MPa mannitol solution. Non‐linear time series analyses methods were used as follows: auto‐correlation and cross‐correlation function, power spectra density function, and complexity of the time series estimated as Approximate Entropy (ApEn).
• Using Approximate Entropy analysis we found that the level of temporal complexity of the electrical signals was affected by the environmental conditions, decreasing when the plant was subjected to a low osmotic potential. Electrical spikes observed only after stimuli followed a power law distribution, which is indicative of scale invariance.
• Our results suggest that changes in complexity of the electrical signals could be associated with water stress conditions in plants. We hypothesised that the power law distribution of the spikes could be explained by a self‐organised critical state (SOC) after osmotic stress.

     

Control of Pratylenchus brachyurus in soybean with Trichoderma spp. and resistance inducers

Trichoderma spp. is a fungus with nematode control potential; however, its potential to control the root lesion nematode Pratylenchus brachyurus remains poorly studied. Thus, the aim of this study was to select Trichoderma spp. isolates and assess their ability to control P. brachyurus in soybean crops. Different experiments were conducted aiming at selecting isolates, assessing whether they were able to reduce nematode penetration in plants or cause mortality in vitro, and whether they were able to induce resistance in soybean, as well as at studying the possibility of using the selected isolates associated with resistance inducers (acibenzolar‐S‐methyl, Ecolife^? and AgroMos^?). The selection experiment found three isolates showing satisfactory results, namely GF422, GF425 and GF427; the GF362 isolate was assessed in the subsequent experiments. These four isolates reduced P. brachyurus penetration in soybean roots and promoted nematode mortality in vitro. Increased total protein and catalase activity were recorded, mainly in the 72‐hr assessments. Overall, the protein production was different between isolates. The best results were found in the combination between the GF362 isolate and the three resistance inducers, between GF427 and Ecolife^?, between GF427 and AgroMos^? and between GF422 and Ecolife^?.     

Quick and accurate detection of Sclerotinia sclerotiorum and Phomopsis spp. in soybean seeds using qPCR and seed‐soaking method

Seed‐borne pathogenic fungi can cause serious damage to soybean crops by reducing the germination, vigour and emergence of the seeds. Special attention should be paid to pathogen detection in seeds to prevent its introduction in disease‐free areas. Considering the importance of rapid and successful diagnosis of seed‐borne pathogenic fungi in soybeans, this study evaluated a method to detect Sclerotinia sclerotiorum and Phomopsis spp. in seeds using quantitative polymerase chain reaction (qPCR). Naturally infested samples were subjected to detection using qPCR and blotter test, and the findings were compared. Using soybean seeds soaked in water, both pathogens were detected at an infestation level up a 0.0625% (one infected seed out of 1,599 healthy seeds) by qPCR. This technique allowed the detection of 300 fg of S. sclerotiorum and 30 fg of Phomopsis spp. DNA in the seed samples. Phomopsis spp. was detected in 40.7% of the evaluated seed batches (81 batches) and S. sclerotiorum was detected in 32.1% of the evaluated batches, although most of the seeds had low infestation levels. It was up to 28.5 times more efficient to use qPCR rather than blotter test to detect pathogens with a low incidence of occurrence in soybean seeds. If routinely used to test healthy seeds, qPCR would contribute to reducing soybean losses due to diseases as well as decreasing the costs required to control those diseases.     

Spatial and temporal patterns of environmental DNA detection to inform sampling protocols in lentic and lotic systems

The development of efficient sampling protocols for the capture of environmental DNA (eDNA) could greatly help improve accuracy of occupancy monitoring for species that are difficult to detect. However, the process of developing a protocol in situ is complicated for rare species by the fact that animal locations are often unknown. We tested sampling designs in lake and stream systems to determine the most effective eDNA sampling protocols for two rare species: the Sierra Nevada yellow‐legged frog (Rana sierrae) and the foothill yellow‐legged frog (Rana boylii). We varied water volume, spatial sampling, and seasonal timing in lakes and streams; in lakes we also tested multiple filter types. We found that filtering 2 L versus 1 L increased the odds of detection in streams 5.42X (95% CI: 3.2–9.19X) in our protocol, from a probability of 0.51–0.85 per technical replicate. Lake sample volumes were limited by filter clogging, and we found no effect of volume or filter type. Sampling later in the season increased the odds of detection in streams by 1.96X for every 30 days (95% CI: 1.3–2.97X) but there was no effect for lakes. Spatial autocorrelation of the quantity of yellow‐legged frog eDNA captured in streams ceased between 100 and 200 m, indicating that sampling at close intervals is important.