Dataset Information

Detection and quantification of Plectosphaerella cucumerina, a potential biological control agent of potato cyst nematodes, by using conventional PCR, real-time PCR, selective media, and baiting.

ABSTRACT: Potato cyst nematodes (PCN) are serious pests in commercial potato production, causing yield losses valued at approximately $300 million in the European Community. The nematophagous fungus Plectosphaerella cucumerina has demonstrated its potential as a biological control agent against PCN populations by reducing field populations by up to 60% in trials. The use of biological control agents in the field requires the development of specific techniques to monitor the release, population size, spread or decline, and pathogenicity against its host. A range of methods have therefore been developed to monitor P. cucumerina. A species-specific PCR primer set (PcCF1-PcCR1) was designed that was able to detect the presence of P. cucumerina in soil, root, and nematode samples. PCR was combined with a bait method to identify P. cucumerina from infected nematode eggs, confirming the parasitic ability of the fungus. A selective medium was adapted to isolate the fungus from root and soil samples and was used to quantify the fungus from field sites. A second P. cucumerina-specific primer set (PcRTF1-PcRTR1) and a Taqman probe (PcRTP1) were designed for real-time PCR quantification of the fungus and provided a very sensitive means of detecting the fungus from soil. PCR, bait, and culture methods were combined to investigate the presence and abundance of P. cucumerina from two field sites in the United Kingdom where PCN populations were naturally declining. All methods enabled differences in the activity of P. cucumerina to be detected, and the results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.

SUBMITTER: Atkins SD

PROVIDER: S-EPMC169141 | biostudies-literature | 2003 Aug

REPOSITORIES: biostudies-literature

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Detection and quantification of Plectosphaerella cucumerina, a potential biological control agent of potato cyst nematodes, by using conventional PCR, real-time PCR, selective media, and baiting.

Atkins S D SD Clark I M IM Sosnowska D D Hirsch P R PR Kerry B R BR

Applied and environmental microbiology 20030801 8

Potato cyst nematodes (PCN) are serious pests in commercial potato production, causing yield losses valued at approximately $300 million in the European Community. The nematophagous fungus Plectosphaerella cucumerina has demonstrated its potential as a biological control agent against PCN populations by reducing field populations by up to 60% in trials. The use of biological control agents in the field requires the development of specific techniques to monitor the release, population size, sprea ...[more]

PMID: 12902272

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Project description:Potato cyst nematodes (PCN), such as Globodera rostochiensis and Globodera pallida, are quarantine restricted pests of potato causing major yield and financial losses to farmers. G. rostochiensis was first reported from Kenya's key potato growing area in 2015. We sought to determine the diversity, prevalence and distribution of PCN species across the country by conducting a country-wide survey between 2016 and 2018, which included a more focused, follow-up assessment in three key potato growing counties. A total of 1,348 soil samples were collected from 20 potato growing counties. Information regarding local potato farming practices, potato cultivar use, their diversity and availability was also recorded. PCN cysts were obtained from 968 samples (71.8%) in all the counties surveyed, with Nyandarua County recording the highest PCN field-incidence at 47.6%. The majority of PCN populations, 99.9%, were identified as G. rostochiensis, while G. pallida was recovered from just one field, in a mixed population with G. rostochiensis. Inconsistencies in PCR amplification efficiency was observed for G. rostochiensis using the recommended EPPO primers, compared with ITS primers AB28/TW81, indicating that this protocol cannot be entirely relied upon to effectively detect PCN. Egg density in Nyandarua County varied between 30.6 and 158.5 viable eggs/g soil, with an average egg viability of 78.9 ± 2.8% (min = 11.6%, max = 99.9%). The PCN-susceptible potato cultivar named Shangi was the most preferred and used by 65% of farmers due to its shorter dormancy and cooking time, while imported cultivars (Destiny, Jelly, Manitou, and Markies) with resistance to G. rostochiensis were used by 7.5% of farmers due to unavailability and/or limited access to seeds. Thus, most farmers preferred using their own farm-saved seeds as opposed to purchasing certified seeds. Establishing the distribution and prevalence of PCN and elucidating the local farming practices that could promote the spread of PCN is a necessary precursor to the implementation of any containment or management strategy in the country and ultimately across the region.

Project description:BackgroundXylanase can replace chemical additives to improve the volume and sensory properties of bread in the baking. Suitable baking xylanase with improved yield will promote the application of xylanase in baking industry. The xylanase XYNZG from the Plectosphaerella cucumerina has been previously characterized by heterologous expression in Pichia pastoris. However, P. pastoris is not a suitable host for xylanase to be used in the baking process since P. pastoris does not have GRAS (Generally Regarded As Safe) status and requires large methanol supplement during the fermentation in most conditions, which is not allowed to be used in the food industry. Kluyveromyces lactis, as another yeast expression host, has a GRAS status, which has been successfully used in food and feed applications. No previous work has been reported concerning the heterologous expression of xylanase gene xynZG in K. lactis with an aim for application in baking.ResultsThe xylanase gene xynZG from the P. cucumerina was heterologously expressed in K. lactis. The recombinant protein XYNZG in K. lactis presented an approximately 19 kDa band on SDS-PAGE and zymograms analysis. Transformant with the highest halo on the plate containing the RBB-xylan (Remazol Brilliant Blue-xylan) was selected for the flask fermentation in different media. The results indicated that the highest activity of 115 U/ml at 72 h was obtained with the YLPU medium. The mass spectrometry analysis suggested that the hydrolytic products of xylan by XYNZG were mainly xylobiose and xylotriose. The results of baking trials indicated that the addition of XYNZG could reduce the kneading time of dough, increase the volume of bread, improve the texture, and have more positive effects on the sensory properties of bread.ConclusionsXylanase XYNZG is successfully expressed in K. lactis, which exhibits the highest activity among the published reports of the xylanase expression in K. lactis. The recombinant XYNZG can be used to improve the volume and sensory properties of bread. Therefore, the expression yield of recombinant XYNZG can be further improved through engineered strain containing high copy numbers of the XYNZG, and optimized fermentation condition, making bread-baking application possible.

Dataset Information

Detection and quantification of Plectosphaerella cucumerina, a potential biological control agent of potato cyst nematodes, by using conventional PCR, real-time PCR, selective media, and baiting.

Publications

Detection and quantification of Plectosphaerella cucumerina, a potential biological control agent of potato cyst nematodes, by using conventional PCR, real-time PCR, selective media, and baiting.

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