Isolation of nucleotide binding site-leucine rich repeat and kinase resistance gene analogues from sugarcane (Saccharum spp.)

BACKGROUND: Resistance gene analogues (RGAs) have been isolated from many crops and offer potential in breeding for disease resistance through marker-assisted selection, either as closely linked or as perfect markers. Many R-gene sequences contain kinase domains, and indeed kinase genes have been reported as being proximal to R-genes, making kinase analogues an additionally promising target. The first step towards utilizing RGAs as markers for disease resistance is isolation and characterization of the sequences. RESULTS: Sugarcane clone US01-1158 was identified as resistant to yellow leaf caused by the sugarcane yellow leaf virus (SCYLV) and moderately resistant to rust caused by Puccinia melanocephala Sydow & Sydow. Degenerate primers that had previously proved useful for isolating RGAs and kinase analogues in wheat and soybean were used to amplify DNA from sugarcane (Saccharum spp.) clone US-01-1158. Sequences generated from 1512 positive clones were assembled into 134 contigs of between two and 105 sequences. Comparison of the contig consensuses with the NCBI sequence database using BLASTx showed that 20 had sequence homology to nuclear binding site and leucine rich repeat (NBS-LRR) RGAs, and eight to kinase genes. Alignment of the deduced amino acid sequences with similar sequences from the NCBI database allowed the identification of several conserved domains. The alignment and resulting phenetic tree showed that many of the sequences had greater similarity to sequences from other species than to one another. CONCLUSION: The use of degenerate primers is a useful method for isolating novel sugarcane RGA and kinase gene analogues. Further studies are needed to evaluate the role of these genes in disease resistance.     

Fungicide seed treatment efficacy against Microdochium nivale and M. majus in vitro and in vivo

BACKGROUND: Seed-borne Microdochium majus (Wollenweber) and M. nivale Fries are the primary pathogens responsible for Fusarium seedling blight in the UK. The two species show differences in pathogenicity, host preference and sensitivities to temperature, but their relative sensitivities to fungicide seed treatments are unknown. The aim was firstly to determine the efficacy of fungicide seed treatments towards single-spore isolates of M. majus and M. nivale using in vitro experiments, and subsequently to determine efficacy in vivo over a range of temperatures. RESULTS: Differences in EC(50) values between all seed treatments were evident from the in vitro experiments and ranged from 0.028 mg L(-1) for fludioxonil to 22.8 mg L(-1) for carboxin + thiram. The two seed treatments that showed best performance in vitro were used to examine efficacy towards seed-borne infection in vivo at 4, 8, 12 and 16 degrees C. Generally, seedling emergence improved and the severity of stem-base disease symptoms on emerged seedlings was reduced for both species through the use of the fungicides. The combination of fludioxonil + difenconazole showed improved performance compared with fludioxonil alone. Significantly less severe symptoms were observed through the use of fludioxonil and fludioxonil + difenconazole compared with bitertanol + fuberidazole at 12 degrees C and for all except one M. nivale infected seed lot at 8 degrees C. CONCLUSIONS: Differences in fungicide sensitivity between the two species in vitro were not evident in vivo. This is the first report of the effect of fungicide seed treatments on the control of seedling blight caused by M. majus and M. nivale.     

Spatiotemporal Distribution of Chinavia hilaris (Hemiptera: Pentatomidae) in Peanut-Cotton Farmscapes

The green stink bug, Chinavia hilaris (Say) (Hemiptera: Pentatomidae), is a pest of cotton in the southeastern United States, but little is known concerning its spatiotemporal distribution in agricultural farmscapes. Therefore, spatiotemporal distribution of C. hilaris in farmscapes where cotton fields adjoined peanut was examined weekly. Spatial patterns of C. hilaris counts were analyzed using SADIE (Spatial Analysis by Distance Indices) methodology. Interpolated maps of C. hilaris density were used to visualize abundance and distribution of C. hilaris in crops. For the six peanut-cotton farmscapes studied, the frequency of C. hilaris in cotton (94.8%) was significantly higher than in peanut (5.2%), and nymphs were rarely detected in peanut, indicating that peanut was not a source of C. hilaris into cotton. Significantly, aggregated spatial distributions were detected in cotton. Maps of local clustering indices depicted patches of C. hilaris in cotton, mainly at field edges including the peanut-to-cotton interface. Black cherry (Prunus serotina Ehrh.) and elderberry (Sambucus nigra subsp. canadensis [L.] R. Bolli) grew in habitats adjacent to crops, C. hilaris were captured in pheromone-baited stink bug traps in these habitats, and in most instances, C. hilaris were observed feeding on black cherry and elderberry in these habitats before colonization of cotton. Spatial distribution of C. hilaris in these farmscapes revealed that C. hilaris colonized cotton field edges near these two noncrop hosts. Altogether, these findings suggest that black cherry and elderberry were sources of C. hilaris into cotton. Factors affecting the spatiotemporal dynamics of C. hilaris in peanut-cotton farmscapes are discussed.     

PCR assays for the sugarcane rust pathogens Puccinia kuehnii and P. melanocephala and detection of a SNP associated with geographical distribution in P. kuehnii

Puccinia kuehnii and P. melanocephala cause orange and brown rust of sugarcane, respectively. Puccinia kuehnii has been confirmed in Asia, Australia and recently, the Caribbean basin, whereas P. melanocephala is distributed among the majority of sugarcane growing regions. Differentiating these two economically significant pathogens visually is problematic and limited to material exhibiting mature disease symptoms or spores. Partial ITS1, ITS2 and complete 5·8S sequences were generated from P. kuehnii and P. melanocephala isolates from around the world. PCR primers and dual labelled hydrolysis probes were designed for each pathogen for use in real‐time PCR and optimized using locked nucleic acids (LNA). The primers amplified DNA from their target pathogens and not from other species of Puccinia or fungal species isolated from sugarcane leaves. Optimized real‐time PCR conditions allowed the detection of 0·19 pg of P. kuehnii or P. melanocephala genomic DNA and differentiated the pathogens on sugarcane leaves prior to observing typical symptoms in the field. Primer‐introduced restriction analysis‐PCR (PIRA‐PCR) was used to detect a single nucleotide polymorphism (Pk ITS1 183A>G) in ITS1 of P. kuehnii. Allele 183A was observed in all samples, whereas 183G was detected in 52% of samples from Asia and Australia yet absent from all Caribbean basin samples. Long distance spore dispersal, dispersal through an intermediate location or improper movement of contaminated material could explain the introduction of P. kuehnii to the Western hemisphere. However, the current proliferation of the pathogen in the Americas is limited to isolates which contain only the 183A allele.     

Toxicity of a formulation of the insecticide indoxacarb to the tarnished plant bug, Lygus lineolaris (Hemiptera: Miridae), and the big-eyed bug, Geocoris punctipes (Hemiptera: Lygaeidae)

Indoxacarb is a new oxadiazine insecticide that has shown outstanding field insecticidal activity. The toxicity of a 145 g litre-1 indoxacarb SC formulation (Steward) was studied on the tarnished plant bug Lygus lineolaris and the big-eyed bug Geocoris punctipes. Both insect species responded very similarly to indoxacarb in topical, tarsal contact and plant feeding toxicity studies. The topical LD50 of the formulation was c 35 ng AI per insect for both species. Prolonged tarsal contact with dry indoxacarb residues did not result in mortality for either insect species. However, both species were susceptible to feeding through dried residues of indoxacarb after spraying on young cotton plants. Feeding on water-washed plants resulted in lower mortality than that observed with unwashed plants, and toxicity declined even more dramatically after a, detergent rinse, indicating that much of the indoxacarb probably resides on the cotton leaf surface or in the waxy cuticle. These results were corroborated by HPLC-mass spectrometry measurements of indoxacarb residues on the plants. Greater mortality for both species was observed in a higher relative humidity environment. Higher levels of accumulated indoxacarb and its active metabolite were detected in dead G punctipes than in L lineolaris after feeding on sprayed, unwashed plants. When female G punctipes ate indoxacarb-treated Heliothis zea eggs, there was significant toxicity. However, only c 15% of the females consumed indoxacarb-treated eggs, and the rest of the females showed a significant diminution of feeding in response to the insecticide. Cotton field studies have shown that indoxacarb treatments at labelled rates lead to a dramatic decline in L lineolaris, with negligible declines in beneficial populations. A major route of intoxication of L lineolaris in indoxacarb-treated cotton fields thus appears to be via oral, and not cuticular, uptake of residues from treated cotton plants. The mechanisms for selectivity/safety for G punctipes are currently under investigation and may be a combination of differential feeding behavior and diminution of feeding by females exposed to indoxacarb-treated eggs.     

Improved Detection of Reproductive Status in Dairy Cows Using Milk Progesterone Measurements

This study tested a model for predicting reproductive status from in-line milk progesterone `measurements. The model is that of Friggens and Chagunda [Theriogenology 64 (2005) 155]. Milk progesterone measurements (n = 55 036) representing 578 lactations from 380 cows were used to test the model. Two types of known oestrus were identified: (1) confirmed oestrus (at which insemination resulted in a confirmed pregnancy, n = 121) and (2) ratified oestrus (where the shape of the progesterone profile matched that of the average progesterone profile of a confirmed oestrus, n = 679). The model detected 99.2% of the confirmed oestruses. This included a number of cases (n = 16) where the smoothed progesterone did not decrease below 4 ng/ml. These cows had significantly greater concentrations of progesterone, both minimum and average, suggesting that between cow variation exists in the absolute level of the progesterone profile. Using ratified oestruses, model sensitivity was 93.3% and specificity was 93.7% for detection of oestrus. Examination of false positives showed that they were largely associated with low concentrations of progesterone, fluctuating around the 4 ng/ml threshold. The distribution of time from insemination until the model detected pregnancy failure had a median of 22 days post-insemination. In this test, the model was run using limited inputs, the potential benefits of including additional non-progesterone information were not evaluated. Despite this, the model performed at least as well as other oestrus detection systems.     

Epidemiological Description of Cystic Ovarian Disease in Argentine Dairy Herds: Risk Factors and Effects on the Reproductive Performance of Lactating Cows

To describe the epidemiology of cystic ovarian disease (COD), to find possible risk factors associated with the incidence of cysts and to analyse the impact of COD on the reproductive performance of dairy cows, databases from 22 dairy herds from the main dairy region in Argentina were retrospectively evaluated throughout a 3‐year period (2009–2011). A total of 248 COD cases over 9156 parturitions were recorded, resulting in a cumulative incidence rate of 2.7%. Cystic ovarian disease incidence density was lower during the first 100 days post‐partum (DPP) than during later stages of lactation. Seasonality had a significant influence on the disease presentation with higher incidence rates during winter and spring. Cows with a previous diagnosis of clinical mastitis showed 2.72 times more chances of developing ovarian cysts. Cystic cows had longer calving to first service and calving to conception intervals and lower conception rate than controls.     

Studies of the interaction between horse chestnut leaf miner (Cameraria ohridella) and bacterial bleeding canker (Pseudomonas syringae pv. aesculi)

The white flowering horse chestnut tree (Aesculus hippocastanum L.) was first introduced to the UK ca. 500 years ago. Over the past eight years however, this tree has suffered from severe attack by a mining insect pest known as the horse chestnut leaf miner (HCLM; Cameraria ohridella) and, concomitantly a gram negative bacterium (Pseudomonas syringae pv. aesculi; Pae). Although studies have investigated the influence of each problem individually on tree growth and vitality the interaction between HCLM and Pae on horse chestnut remains unknown. For this reason four year old horse chestnut seedlings were artificially inoculated with Pae in the presence and absence of HCLM. Effects on tree vitality were assessed by monitoring alterations to leaf chlorophyll content, leaf chlorophyll fluorescence (F_v/F_m ratios) as well as key defensive enzymatic activity (β-1,3-glucanase, peroxidase) of woody tissue at the site of Pae infection. With respect to mean lesion size, the main proxy of Pae success or aggressiveness, lesion size was significantly increased in the presence of HCLM compared to trees inoculated with Pae but where HCLM was controlled using insecticide sprays. Results of this study also indicate that suppression of two key defensive enzymes, β-1,3-glucanase and peroxidase, within woody tissue at the site of Pae infection, possibly as a result of reduced photosynthetic productivity caused by HCLM defoliation, maybe the underlying reason as to why Pae severity is greater in the presence of HCLM.     

Physical barriers for suppression of movement of adult stink bugs into cotton

Nezara viridula (L.), Euschistus servus (Say), and Chinavia hilaris (Say) (Heteroptera: Pentatomidae) are economic pests of cotton in the southeastern USA. Because adult stink bugs exhibit edge-mediated dispersal at crop-to-crop interfaces as they colonize cotton, strategic placement of physical barriers at these interfaces could manage these pests. The objective of this study was to determine the effectiveness of a physical barrier, either synthetic or plant-based, at the peanut-to-cotton interface for suppressing stink bugs that would move to cotton. In 2012 and 2013, sorghum sudangrass (2.4 and 2.1 m high, respectively) was significantly taller than cotton (1.4 and 1.3 m high, respectively) which was taller than peanut (0.4 and 0.5 m high, respectively). Buckwheat (0.6 m high), planted only in 2012, was significantly taller than peanut, but shorter than cotton. For both years of the study, sorghum sudangrass and a 1.8-m-high polypropylene barrier wall effectively deterred dispersal of stink bugs into cotton. Because each of these barriers was taller than cotton, their success in protecting cotton likely was due to disruption of the flight of stink bugs from low-growing peanut into cotton. The shortest barrier wall (0.6-m-high) did not suppress stink bug dispersal into cotton probably because it was approximately the same height as peanut. In 2012, flowering buckwheat increased the efficacy of Trichopoda pennipes (F.) attacking N. viridula in cotton although it did not deter dispersal of stink bugs. In conclusion, a barrier at least as tall as cotton can effectively retard the entry of stink bug adults into cotton.