Factors impacting the detection of weed seed contaminants in seed lots.

BACKGROUND: The setting and following of phytosanitary standards for weed seeds can lessen the impacts of weeds on agriculture. Standards adopted by seed companies, laboratories and regulators ensure the contamination rates do not exceed some thresholds. Globally sample size standards are set based on the amount needed to obtain a contaminant in a random sample of the seed lot, not detectability. New Zealand requires a 95% confidence that the maximum pest limit of 0.01% of quarantine weed seed contamination is not exceeded in an imported seed lot. We examined 24 samples each containing approximately 150 000 seeds of either perennial ryegrass (12 samples) or white clover seeds (12 samples) that were then spiked with seeds (contaminants) from 12 non-crop species (3-8 seeds of each). We considered factors that may impact detection rates: shape, color, size, and texture relative to the crop, and technician (including a commercial seed laboratory). RESULTS: A linear mixed model fitted to the data indicated significant observer, crop, and seed color, shape, and size effects on detection. Detectability increased by 20% ± 7.7 (± standard error) when seeds had a distinct shape or color (28% ± 8.1), or were larger (23% ± 8.7) rather than smaller, relative to the crop. Commercial laboratory identifications were usually correct at the level of genus, and species for common weeds, but some misidentifications occurred. CONCLUSION: Sample sizes for border inspections should be based on detectability of regulated weed seeds in the crop in combination with weed risk for the crop and location. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Stepping up to the thermogradient plate: a data framework for predicting seed germination under climate change.

BACKGROUND AND AIMS: Seed germination is strongly influenced by environmental temperatures. With global temperatures predicted to rise, the timing of germination for thousands of plant species could change, leading to potential decreases in fitness and ecosystem-wide impacts. The thermogradient plate (TGP) is a powerful but underutilized research tool that tests germination under a broad range of constant and alternating temperatures, giving researchers the ability to predict germination characteristics using current and future climates. Previously, limitations surrounding experimental design and data analysis methods have discouraged its use in seed biology research. METHODS: Here, we have developed a freely available R script that uses TGP data to analyse seed germination responses to temperature. We illustrate this analysis framework using three example species: Wollemia nobilis, Callitris baileyi and Alectryon subdentatus. The script generates >40 germination indices including germination rates and final germination across each cell of the TGP. These indices are then used to populate generalized additive models and predict germination under current and future monthly maximum and minimum temperatures anywhere on the globe. KEY RESULTS: In our study species, modelled data were highly correlated with observed data, allowing confident predictions of monthly germination patterns for current and future climates. Wollemia nobilis germinated across a broad range of temperatures and was relatively unaffected by predicted future temperatures. In contrast, C. baileyi and A. subdentatus showed strong seasonal temperature responses, and the timing for peak germination was predicted to shift seasonally under future temperatures. CONCLUSIONS: Our experimental workflow is a leap forward in the analysis of TGP experiments, increasing its many potential benefits, thereby improving research predictions and providing substantial information to inform management and conservation of plant species globally.

Rapid and Sensitive Detection of Tomato Brown Rugose Fruit Virus in Tomato and Pepper Seeds by Reverse Transcription Loop-Mediated Isothermal Amplification Assays (Real Time and Visual) and Comparison With RT-PCR End-Point and RT-qPCR Methods.

Tomato brown rugose fruit virus (ToBRFV) represents an emerging viral threat to the productivity of tomato and pepper protected cultivation worldwide. This virus has got the status of quarantine organism in the European Union (EU) countries. In particular, tomato and pepper seeds will need to be free of ToBRFV before entering the EU and before coming on the market. Thus, lab tests are needed. Here, we develop and validate a one-step reverse transcription LAMP platform for the detection of ToBRFV in tomato and pepper leaves, by real-time assay [reverse transcription loop-mediated isothermal amplification (RT-LAMP)] and visual screening (visual RT-LAMP). Moreover, these methods can also be applied successfully for ToBRFV detection in tomato and pepper seeds. The diagnostic specificity and sensitivity of both RT-LAMP and visual RT-LAMP are both 100%, with a detection limit of nearly 2.25 fg/µl, showing the same sensitivity as RT-qPCR Sybr Green, but 100 times more sensitive than end-point RT-PCR diagnostic methods. In artificially contaminated seeds, the proposed LAMP assays detected ToBRFV in 100% of contaminated seed lots, for up to 0.025-0.033% contamination rates in tomato and pepper, respectively. Our results demonstrate that the proposed LAMP assays are simple, inexpensive, and sensitive enough for the detection of ToBRFV, especially in seed health testing. Hence, these methods have great potential application in the routine detection of ToBRFV, both in seeds and plants, reducing the risk of epidemics.

A PCR, qPCR, and LAMP Toolkit for the Detection of the Wheat Blast Pathogen in Seeds.

Wheat blast is a devastating disease caused by the pathogenic fungus Pyricularia oryzae. Wheat blast first emerged in South America before more recently reaching Bangladesh. Even though the pathogen can spread locally by air-dispersed spores, long-distance spread is likely to occur via infected wheat seed or grain. Wheat blast epidemics are caused by a genetic lineage of the fungus, called the Triticum lineage, only differing from the other P. oryzae lineages by less than 1% genetic divergence. In order to prevent further spread of this pathogen to other wheat-growing areas in the world, sensitive and specific detection tools are needed to test for contamination of traded seed lots by the P. oryzae Triticum lineage. In this study, we adopted a comparative genomics approach to identify new loci specific to the P. oryzae Triticum lineage and used them to design a set of new markers that can be used in conventional polymerase chain reaction (PCR), real-time PCR, or loop-mediated isothermal amplification (LAMP) for the detection of the pathogen, with improved inclusivity and specificity compared to currently available tests. A preliminary biological enrichment step of the seeds was shown to improve the sensitivity of the tests, which enabled the detection of the target at an infection rate as low as 0.25%. Combined with others, this new toolkit may be particularly beneficial in preventing the trade of contaminated seeds and in limiting the spread of the disease.

A comparative study of the thermal ranges of three germination criteria of a tropical tree with bioeconomic interest: Carapa surinamensis Miq. (Meliaceae) / Estudo comparativo sobre a variação térmica em três critérios de germinação de uma árvore tropical com interesse econômico: Carapa surinamensis Miq. (Meliaceae)

Abstract Species of the Carapa spp. complex, occurring in the Neotropics, Africa and India, have multiple uses, including timber, with the seed oil being used in phyto-pharmaceutical products and cosmetics. This study aimed to determine the thermal ranges of the germination process, comparing germination criteria used by seed physiologists and seed technologists, and to suggest recommendations for seed quality assessment. Germination was assessed at constant temperatures between 10 ─ 40 °C using three germination criteria: (1) radicle length ≥ 0.5 cm (physiological criterion); (2) epicotyl length ≥ 1 cm; and (3) epicotyl length ≥ 5 cm (criterion for seed quality tests). The base temperature was similar for the three criteria and ranged between 10 ─ 2 °C. The Maguire's Speed Index indicated 30 °C as most adequate. However, the upper temperature limit differed: for radicle protrusion it was above 40 ºC; and for both epicotyl lengths, it was between 35 ─ 40 °C. Seed coat removal accelerated the germination process of these recalcitrant seeds, and is recommended for seed quality assessment, which allows completion of the germination trial in approximately one month.

Resumo As espécies do complexo Carapa spp. ocorrem na região Neotropical, na África e na Índia, têm usos múltiplos, fornece madeira de valor comercial e o óleo extraído das sementes tem uso fitoterápico e cosmético. O objetivo deste trabalho foi determinar a faixa térmica tolerável do processo germinativo, comparando os critérios de germinação utilizados pelos fisiologistas e os tecnólogos de sementes, e sugerir recomendações para a avaliação da qualidade das sementes. A germinação foi avaliada em temperaturas constantes entre 10 e 40 °C utilizando três critérios de germinação: (1) formação da radícula ≥ 0,5 cm (critério fisiológico); (2) alongamento de epicótilo ≥ 1 cm; e (3) alongamento de epicótilo ≥ 5 cm (critério para testes de qualidade de sementes). A temperatura de base foi semelhante para os três critérios entre 10 e 12 °C. O índice de velocidade de Maguire indicou 30 °C como a temperatura mais adequada. O limite superior de temperatura diferiu entre os critérios, sendo acima de 40 ºC para protrusão da radícula e para ambos os alongamentos de epicótilo entre 35 e 40 °C. A remoção do tegumento de semente acelerou o processo de germinação dessas sementes recalcitrantes sendo recomendada para a avaliação da qualidade da semente, o que permite concluir o teste de germinação em aproximadamente um mês.

Detection of Tilletia caries, Tilletia laevis and Tilletia controversa wheat grain contamination using loop-mediated isothermal DNA amplification (LAMP).

The study describes a novel diagnostic protocol based on a loop-mediated isothermal DNA amplification (LAMP) for identification of wheat grains infection by Tilletia laevis, Tilletia caries (common bunt) and Tilletia controversa (draft bunt). The presented data showed that the LAMP analysis is a simple, specific and rapid method that could be used for detection of Tilletia spp. in contaminated grain samples. The lowest DNA concentration required for the successful detection of Tilletia spp. strains were estimated to be 0.001 ng/µl. Simultaneously the detection limit for wheat grain contamination by T. caries and T. laevis teliospores was estimated at 20 µg per 100 g of grain. For T. controversa detection limit was lower and was approximately 20 mg of teliospores per 100 g of grain. The negative results of the LAMP reactions were achieved for the most common fungal species colonizing wheat grain like Fusarium spp., Alternaria sp., Cladosporium sp., Helminthosporium sp., and Penicillium sp.

A Spectrophotometric Assay for Robust Viability Testing of Seed Batches Using 2,3,5-Triphenyl Tetrazolium Chloride: Using Hordeum vulgare L. as a Model.

A comparative analysis was carried out of published methods to assess seed viability using 2,3,5-triphenyltetrazolium chloride (TTC) based assays of seed batches. The tests were carried out on seeds of barley (Hordeum vulgare cv. Optic) as a model. We established that 10% [w/v] trichloroacetic acid (TCA)/methanol is superior to the acetone and methanol-only based methods: allowing the highest recovery of formazan and the lowest background optical density (OD) readings, across seed lots comprising different ratios of viable and dead seeds. The method allowed a linear-model to accurately capture the statistically significant relationship between the quantity of formazan that could be extracted using the method we developed and the seed temperature-response, and seed viability as a function of artificially aged seed lots. Other quality control steps are defined to help ensure the assay is robust and these are reported in a Standard Operating Procedure.

Ex-post assessment of genetically modified, low level presence in Canadian flax.

Canada is the world's largest producer and exporter of flaxseed. In 2009, DNA from deregistered genetically modified (GM) CDC Triffid was detected in a shipment of Canadian flaxseed exported to Europe, causing a large decrease in the amount of flax planted in Canada and a major shift in export markets. The flax industry in Canada undertook major changes to ensure the removal of transgenic flax from the supply chain. To demonstrate compliance, Canada adopted a protocol involving testing grain samples (post-harvest) using an RT-PCR test for the construct found in CDC Triffid. Efforts to remove the presence of GM flax from the value chain included reconstituting major flax varieties from GM-free plants. The reconstituted varieties represented the majority of planting seed in 2014. This study re-evaluates GM flax presence in Canadian grain stocks for an updated dataset (2009-2015) using a previously described simulation model to estimate low-level GM presence. Additionally, losses to the Canadian economy resulting from the reduction in flax production and export opportunities, costs associated with reconstituting major flax varieties, and testing for the presence of GM flax along the flax value chain are estimated.

A comparative study of the thermal ranges of three germination criteria of a tropical tree with bioeconomic interest: Carapa surinamensis Miq. (Meliaceae)

Abstract Species of the Carapa spp. complex, occurring in the Neotropics, Africa and India, have multiple uses, including timber, with the seed oil being used in phyto-pharmaceutical products and cosmetics. This study aimed to determine the thermal ranges of the germination process, comparing germination criteria used by seed physiologists and seed technologists, and to suggest recommendations for seed quality assessment. Germination was assessed at constant temperatures between 10 40 °C using three germination criteria: (1) radicle length 0.5 cm (physiological criterion); (2) epicotyl length 1 cm; and (3) epicotyl length 5 cm (criterion for seed quality tests). The base temperature was similar for the three criteria and ranged between 10 2 °C. The Maguires Speed Index indicated 30 °C as most adequate. However, the upper temperature limit differed: for radicle protrusion it was above 40 ºC; and for both epicotyl lengths, it was between 35 40 °C. Seed coat removal accelerated the germination process of these recalcitrant seeds, and is recommended for seed quality assessment, which allows completion of the germination trial in approximately one month.

Resumo As espécies do complexo Carapa spp. ocorrem na região Neotropical, na África e na Índia, têm usos múltiplos, fornece madeira de valor comercial e o óleo extraído das sementes tem uso fitoterápico e cosmético. O objetivo deste trabalho foi determinar a faixa térmica tolerável do processo germinativo, comparando os critérios de germinação utilizados pelos fisiologistas e os tecnólogos de sementes, e sugerir recomendações para a avaliação da qualidade das sementes. A germinação foi avaliada em temperaturas constantes entre 10 e 40 °C utilizando três critérios de germinação: (1) formação da radícula 0,5 cm (critério fisiológico); (2) alongamento de epicótilo 1 cm; e (3) alongamento de epicótilo 5 cm (critério para testes de qualidade de sementes). A temperatura de base foi semelhante para os três critérios entre 10 e 12 °C. O índice de velocidade de Maguire indicou 30 °C como a temperatura mais adequada. O limite superior de temperatura diferiu entre os critérios, sendo acima de 40 ºC para protrusão da radícula e para ambos os alongamentos de epicótilo entre 35 e 40 °C. A remoção do tegumento de semente acelerou o processo de germinação dessas sementes recalcitrantes sendo recomendada para a avaliação da qualidade da semente, o que permite concluir o teste de germinação em aproximadamente um mês.

Seed quality test methods of Marsdenia tenacissima / 中草药

Objective: To optimize the testing methods for seed quality of Marsdenia tenacissima, and provide basis for establishing seed quality standard of M. tenacissima. Methods: The seed quality of M. tenacissima from different producing areas was measured based on the International Seed Testing Protocol made up by ISTA and Rules for Agricultural Seed Testing issued by China. Results: The samples weight of M. tenacissima were at least 900 g for purity analysis and were at least 90 g for testing. The 1 000-seed weight was determined by 500-seed method, and the water content was carried out by higher temperature (133 ± 2)℃ for 6 h. After soaking in water for 24 h, M. tenacissima seeds were cultured in wet sand at 30℃ for 1-8 d under illumination for germination testing. Seed viability was tested by TTC method in 35℃ for 3 h. Conclusion: The seed testing methods for quality items of M. tenacissima have been initially established.