Phytophthora capsici, the causal agent of Phytophthora blight, is a prominent and economically damaging oomycete pathogen in South Georgia. P. capsici causes crown, root, leaf, stem, and fruit infections on a wide range of vegetable crops. Oomycete pathogens such as P. capsici are dispersed in water, as their zoospores are flagellated and can move through runoff. Irrigation ponds are often reservoirs for different pathogens, and reusing the captured runoff is increasing in popularity to decrease irrigation costs. This combination allows for unintended outbreaks of diseases by pumping the contaminated runoff onto susceptible crops. Detection and identification of these pathogens are crucial steps in disease management, and rapid detection can ensure timely application of disease control measures. In this study, 42 irrigation ponds in nine counties from South Georgia were surveyed for the presence of P. capsici using a novel filtration method in conjunction with a LAMP assay specific for P. capsici. Ten ponds in five counties were found to have P. capsici as detected from the assay, suggesting that testing of irrigation ponds for P. capsici and other pathogens should be conducted to assist in preventing disease outbreaks.
A loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of Cucurbit leaf crumple virus (CuLCrV), one of the most important begomoviruses that infects cucurbits worldwide. A set of six specific primers targeting a total 240 nt sequence regions in the DNA A of CuLCrV were designed and synthesized for detection of CuLCrV from infected leaf tissues using real-time LAMP amplification with the Genie® III system, which was further confirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. The optimum reaction temperature and time were determined, and no cross-reactivity was seen with other begomoviruses. The LAMP assay could amplify CuLCrV from a mixed virus assay. The sensitivity assay demonstrated that the LAMP reaction was more sensitive than conventional PCR, but less sensitive than qPCR. However, it was simpler and faster than the other assays evaluated. The LAMP assay also amplified CuLCrV-infected symptomatic and asymptomatic samples more efficiently than PCR. Successful LAMP amplification was observed in mixed virus-infected field samples. This simple, rapid, and sensitive method has the capacity to detect CuLCrV in samples collected in the field and is therefore suitable for early detection of the disease to reduce the risk of epidemics.
Apples grown in high heat, high light, and low humidity environments are at risk for sun injury disorders like sunburn and associated crop losses. Understanding the physiological and molecular mechanisms underlying sunburn will support improvement of mitigation strategies and breeding for more resilient varieties. Numerous studies have highlighted key biochemical processes involved in sun injury, such as the phenylpropanoid and reactive oxygen species (ROS) pathways, demonstrating both enzyme activities and expression of related genes in response to sunburn conditions. Most previous studies have focused on at-harvest activity of a small number of genes in response to heat stress. Thus, it remains unclear how stress events earlier in the season affect physiology and gene expression. Here, we applied heat stress to mid-season apples in the field and collected tissue along a time course-24, 48, and 72 h following a heat stimulus-to investigate dynamic gene expression changes using a transcriptomic lens. We found a relatively small number of differentially expressed genes (DEGs) and enriched functional terms in response to heat treatments. Only a few of these belonged to pathways previously described to be involved in sunburn, such as the AsA-GSH pathway, while most DEGs had not yet been implicated in sunburn or heat stress in pome fruit.
This prospective study was undertaken in Dhaka Shishu Hospital from 15th March 2005 to 15th October 2006 to evaluate the role of simple hematological test for early diagnosis of neonatal sepsis. Eighty suspected cases of septicaemia admitted in neonatal ward of Dhaka Shishu Hospital were included in this study. Patients those who had history of perinatal asphyxia, infant of diabetic mother, congenital cyanotic heart disease etc. were excluded from the study. Thirty neonates without sign symptoms of septicaemia admitted for other causes like jaundice, feeding problems etc. were taken as a control group. After taking informed consent data were collected in structured questionnaire. Following laboratory investigations like total WBC count, differential count, absolute neutrophil count, band cell count, platelet count, CRP, blood culture were done. Micro-ESR was done as bed side test. Immature and total neutrophil ratio (I/T ratio) detected from total neutrophil and band form count. Patients with positive blood culture were categorized as definite sepsis. Patients with negative blood culture but abnormal hematological report suggestive of sepsis were categorized as probable sepsis. Those who had no signs of sepsis were categorized as control group. Absolute neutrophil count (ANC) had low sensitivity (13%) but Micro-ESR, CRP, I/T ratio, platelet count had moderately high sensitivity and specificity. These simple hematological screen are useful marker for early diagnosis of neonatal sepsis.
Huanglongbing (HLB) or citrus greening disease is the most devastating disease of citrus worldwide. This disease, caused by the bacterium ‘Candidatus Liberibacter asiaticus’ (CLas), leads to low fruit quality and unproductive trees. In 2008, HLB was found in a residential citrus tree in Savannah, Georgia, and, as a result, the state has been quarantined for this disease since 2009. Nonetheless, little is known about the distribution of CLas within Georgia, even though the commercial planting of citrus in Georgia has increased exponentially in recent years. In 2019, 94 samples from commercial and residential citrus trees within 11 counties in coastal and southern Georgia were collected and tested for the presence of CLas. Molecular testing results revealed the presence of CLas in three counties where HLB had not been previously reported and in 9% of samples overall. This is the first definitive report confirming HLB in southern Georgia counties besides those along the coast.
Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in vegetable production annually. Phytophthora capsici is soil-borne and a persistent problem in vegetable fields due to its long-lived survival structures (oospores and chlamydospores) that resist weathering and degradation. The main method of dispersal is through the production of zoospores, which are single-celled, flagellated spores that can swim through thin films of water present on surfaces or in water-filled soil pores and can accumulate in puddles and ponds. Therefore, irrigation ponds can be a source of the pathogen and initial points of disease outbreaks. Detection of P. capsici in irrigation water is difficult using traditional culture-based methods because other microorganisms present in the environment, such as Pythium spp., usually overgrow P. capsici making it undetectable. To determine the presence of P. capsici spores in water sources (irrigation water, runoff, etc.), we developed a hand pump-based filter paper (8-10 µm) method that captures the pathogen's spores (zoospores) and is later used to amplify the pathogen's DNA through a novel loop-mediated isothermal amplification (LAMP) assay designed for the specific amplification of P. capsici. This method can amplify and detect DNA from a concentration as low as 1.2 x 102 zoospores/mL, which is 40 times more sensitive than conventional PCR. No cross-amplification was obtained when testing closely related species. LAMP was also performed using a colorimetric LAMP master mix dye, displaying results that could be read with the naked eye for on-site rapid detection. This protocol could be adapted to other pathogens that reside, accumulate, or are dispersed via contaminated irrigation systems.
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.
