Bacterial leaf scorch, caused by Xylella fastidiosa , is a major threat to blueberry production in the southeastern United States. Management of this devastating disease is challenging and often requires early detection of the pathogen to reduce major loss. There are several different molecular and serological detection methods available to identify the pathogen. Knowing the efficiency and suitability of these detection techniques for application in both field and laboratory conditions is important when selecting the appropriate detection tool. Here, we compared the efficiency and the functionality of four different molecular detection techniques (PCR, real-time PCR, LAMP and AmplifyRP® Acceler8™) and one serological detection technique (DAS-ELISA). The most sensitive method was found to be real-time PCR with the detection limit of 25 fg of DNA molecules per reaction (≈9 genome copies), followed by LAMP at 250 fg per reaction (≈90 copies), AmplifyRP® Acceler8™ at 1 pg per reaction (≈350 copies), conventional PCR with nearly 1.25 pg per reaction (≈ 440 copies) and DAS-ELISA with 1x10 5 cfu/mL of Xylella fastidiosa . Validation between assays with 10 experimental samples gave consistent results beyond the variation of the detection limit. Considering robustness, portability, and cost, LAMP and AmplifyRP® Acceler8™ were not only the fastest methods but also portable to the field and didn’t require any skilled labor to carry out. Among those two, AmplifyRP® Acceler8™ was faster but more expensive and less sensitive than LAMP. On the other hand, real-time PCR was the most sensitive assay and required comparatively lesser time than C-PCR and DAS-ELISA, which were the least sensitive assays in this study, but all three assays are not portable and needed skilled labor to proceed. These findings should enable growers, agents, and diagnosticians to make informed decisions regarding the selection of an appropriate diagnostic tool for X . fastidiosa on blueberry.
Fusarium wilt of watermelon, caused by Fusarium oxysporum f. sp. niveum (FON), is pathogenic only to watermelon and has become one of the main limiting factors in watermelon production internationally. Detection methods for this pathogen are limited, with few published molecular assays available to differentiate FON from other formae speciales of F. oxysporum. FON has four known races that vary in virulence but are difficult and costly to differentiate using traditional inoculation methods and only race 2 can be differentiated molecularly. In this study, genomic and chromosomal comparisons facilitated the development of a conventional polymerase chain reaction (PCR) assay that could differentiate race 3 from races 1 and 2, and by using two other published PCR markers in unison with the new marker, the three races could be differentiated. The new PCR marker, FNR3-F/FNR3-R, amplified a 511 bp region on the “pathogenicity chromosome” of the FON genome that is absent in race 3. FNR3-F/FNR3-R detected genomic DNA down to 2.0 pg/µL. This marker, along with two previously published FON markers, was successfully applied to test over 160 pathogenic FON isolates from Florida, Georgia, and South Carolina. Together, these three FON primer sets worked well for differentiating races 1, 2, and 3 of FON. For each marker, a greater proportion (60 to 90%) of molecular results agreed with the traditional bioassay method of race differentiation compared to those that did not. The new PCR marker should be useful to differentiate FON races and improve Fusarium wilt research.
Here, we report the draft genome sequences of three Fusarium oxysporum f. sp. niveum isolates that were used to design markers for molecular race differentiation. The isolates were collected from watermelon fields in Georgia (USA) and were determined to be different races of F. oxysporum f. sp. niveum using a traditional bioassay.
Fusarium wilt of watermelon (Citrullus lanatus) caused by Fusarium oxysporum f. sp. niveum (Fon), has become an increasing concern of farmers in the southeastern USA, especially in Florida. Management of this disease, most often through the use of resistant cultivars and crop rotation, requires an accurate understanding of an area’s pathogen population structure and phenotypic characteristics. This study improved the understanding of the state’s pathogen population by completing multilocus sequence analysis (MLSA) of two housekeeping genes (BT and TEF) and two loci (ITS and IGS), aggressiveness and race-determining bioassays on 72 isolates collected between 2011 and 2015 from major watermelon production areas in North, Central, and South Florida. Multilocus sequence analysis (MLSA) failed to group race 3 isolates into a single large clade; moreover, clade membership was not apparently correlated with aggressiveness (which varied both within and between clades), and only slightly with sampling location. The failure of multilocus sequence analysis using four highly conserved housekeeping genes and loci to clearly group and delineate known Fon races provides justification for future whole genome sequencing efforts whose more robust genomic comparisons will provide higher resolution of intra-species genetic distinctions. Consequently, these results suggest that identification of Fon isolates by race determination alone may fail to detect economically important phenotypic characteristics such as aggressiveness leading to inaccurate risk assessment.
Anthracnose fruit rot disease, caused by Colletotrichum spp., is the most significant disease problem of commercial strawberry (Fragaria × ananassa) production in the southeastern United States. The hot, humid weather and continuous rainfall in Georgia make Colletotrichum-induced fruit rot a widespread problem in strawberry production. In order to control this disease, growers mainly rely on preventive fungicide applications from flower bud emergence to harvest. The most commonly used single-site fungicides are quinone outside inhibitors (QoIs); the QoI active ingredients azoxystrobin and pyraclostrobin are utilized to manage anthracnose fruit rot. In 2019, we collected 108 strawberry fruits with visible rot symptoms from seven different strawberry farms in Georgia. These farms had received multiple applications of QoI fungicides during the 2019 growing season, as well as in previous seasons. Sensitivities to pyraclostrobin were assessed on 1% malt extract agar using a mycelial growth inhibition assay. Our results demonstrated that a majority of Colletotrichum isolates collected in 2019 were not inhibited by pyraclostrobin, suggesting a growing resistance issue with the QoI fungicides. A PCR-restriction fragment length polymorphism assay showed the presence of the G143A mutation in all QoI “resistant” C. acutatum isolates and none for isolates labeled “reduced sensitivity” or “sensitive”. These results further prove that C. acutatum isolates with the G143A mutation are highly resistant to the QoI fungicide. These findings suggest that there is a high risk of resistance development associated with using pyraclostrobin (likely all QoIs) for controlling anthracnose fruit rot of strawberry in Georgia.
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