Aims: Bacillus licheniformis MZK-05 is a keratinolytic bacterium having potential in dehairing of leather and feather hydrolysis.The present study aimed at to improving the production level of keratinase through gene cloning and expression of recombinant keratinase.Methodology and results: Bacillus licheniformis MZK-05 produced an amplicon of 1,156 bp in a polymerase chain reaction while targeting the gene, kerA, responsible for the enzyme keratinase.The amplicon was subsequently cloned into the plasmid vector pGEX-6p-2 for expression in Escherichia coli BL21.A 58 kD GST-KerA fusion protein was expressed upon IPTG induction which was eventually cleaved by PreScission protease that produced a 39 kD protein.A corresponding increase in proteolytic (312 U/mL) and keratinolytic (196 U/mL) activity were observed with the expressed keratinase.Specific enzyme activities for protease and keratinase, an indication of efficiency of the enzyme, were 2621.84U/mg and 1647 U/mg, respectively and the specific keratinase activity was the highest activity ever reported by any recombinant bacterial strain.Conclusion, significance and impact study: Since the production of keratinase by wild type strain is limited to a certain level, the industrial need could be met by improving the production level through gene cloning and expression of recombinant keratinase.In this connection, the cloning of kerA gene from B. licheniformis MZK-05 into pGEX-6p-2 vector, its expression in Escherichia coli BL21 host and prediction of 3-D model of the expressed protein were performed which will be the basis for industrial production of keratinase in Bangladesh.
Aspergillus flavus infects peanuts and produces a mycotoxin called aflatoxin, a potent human carcinogen. In infected peanuts, it can also affect peanut seed quality by causing seed rot and reducing seed viability, resulting in low germination. In 2020, peanut seeds in Georgia had lower than expected germination and a high frequency of A. flavus contamination. A total of 76 Aspergillus isolates were collected from seven seed lots and their identity and in vitro reaction to QoI (quinone outside inhibitor) fungicide (azoxystrobin) were studied. The isolates were confirmed as A. flavus by morphological characteristics and a PCR (polymerase chain reaction)-based method using species-specific primers. In vitro, these isolates were tested for sensitivity to azoxystrobin. The mean EC50 values ranged from 0.12 to 297.22 μg/mL, suggesting that some isolates were resistant or tolerate to this fungicide. The sequences of cytochrome b gene from these isolates were compared and a single nucleotide mutation (36.8% isolates) was found as Cyt B G143A, which was associated with the total resistance to the QoIs. Another single mutation (15.8% isolates) was also observed as Cyt B F129L, which had been documented for QoI resistance. Therefore, a new major single mutation was detected in the A. flavus natural population in this study, and it might explain the cause of the bad seed quality in 2020. The high frequency of this new single nucleotide mutation exists in the natural population of A. flavus and results in the ineffectiveness of using azoxystrobin seed treatment. New seed treatment fungicides are needed.
In recent years, citrus production has rapidly increased within the state of Georgia (USA), and there are now citrus plantings within at least 32 counties in residential, production, and nursery settings. Among the pathogens capable of infecting citrus are viroids, the smallest plant pathogens. Viroids are comprised of circular, single-stranded RNA ranging from 246-463 nucleotides in length (Ito et al., 2002). Hop stunt viroid (HSVd) is one of several viroids known to infect citrus. This viroid has been previously reported within Arizona, California, Florida, Texas, and Washington in the United States and in other locations throughout the world (Hadidi, 2017). HSVd is often spread mechanically on contaminated tools or through grafting. With a wide host range that includes the families Moraceae, Rosaceae, and Rutaceae (citrus), this viroid can easily move throughout a nursery and spread to other plants (Hadidi, 2017). Symptoms of HSVd include a discoloration and gumming of phloem tissues, stem pitting, bark splitting, and chlorotic and stunted growth in susceptible citrus varieties including tangerines and their hybrids (Hadidi, 2017). There are not typically symptoms on leaves or fruits; however, lime plants have shown some yellowing on leaves (Hadidi, 2017). In May and June of 2020, leaf samples were collected from 12 different citrus plants in nursery settings in Berrien and Mitchell counties in Georgia. The cultivars sampled from Citrus reticulata 'Dekopon'. The sampled trees looked relatively healthy with little or no signs of damage, but were selected for testing to ensure that they were viroid free. Reverse transcription-polymerase chain reaction (RT-PCR) was initially used to verify infection with HSVd. Genomic RNA was extracted from the leaf tissue of twelve plants using the TRIzol reagent (Thermofisher, Waltham, MA). Following cDNA synthesis, samples were tested for the presence of HSVd using the primer pair HSVd-F (5'-GGCAACTCTTCTCAGAATCCAGC-3') and HSVd-R (5'-CCGGGGCTCCTTTCTCAGGTAAGT-3') which produces a 302 bp amplicon (Sano et al., 1988). The PCR reactions for nine of the tested samples did not result in the production of any bands, however the other three samples, all Citrus reticulata 'Dekopon', produced the expected amplicon for HSVd. The amplified products were sequenced using Sanger sequencing (Retrogen Inc, San Diego, CA, USA) and the identity of the fragment sequences was confirmed using BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Partial sequences from these amplicons (deposited as accession number MT632007) shared 99% identity to corresponding HSVd sequences in Genbank (accession number MG779542). In addition to RT-PCR and sequencing, the recombinase-polymerase-amplification (RPA) technology based AmplifyRP® Acceler8™ end-point detection assay (Agdia® Inc., Elkhart, IN) was performed on previously confirmed tissue according to the manufacturer's instructions. This assay also confirmed the presence of HSVd viroid in the three samples that had been previously confirmed via RT-PCR. To the best of our knowledge, this is the first report of HSVd infecting Citrus reticulata 'Dekopon' in Georgia. If this viroid were to spread within the growing Georgia citrus industry, it could pose a significant threat to citrus plantings that contain susceptible varieties. Nursery stock infected with this viroid should be destroyed, and Georgia nursery producers and citrus growers should take appropriate precautions to prevent the spread of this viroid disease, including properly sanitizing tools used for citrus grafting and pruning. Further research is needed to determine the distribution of HSVd and its potential to impact commercial citrus production in Georgia.
The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune cells, and stroma. All of these heterogeneous cells contribute to intestinal homeostasis and participate in antimicrobial host defense. Therefore, identifying a surrogate model for studying immune response and antimicrobial activity of the intestine in an in vitro setting is extremely challenging. In vitro studies using immortalized intestinal epithelial cell lines or even primary crypt organoid culture do not represent the exact physiology of normal intestine and its microenvironment. Here, we discuss a method of culturing mouse colon tissue in a culture dish and how this ex vivo organ culture system can be implemented in studies related to antimicrobial host defense responses. In representative experiments, we showed that colons in organ culture express antimicrobial peptides in response to exogenous IL-1β and IL-18. Further, the antimicrobial effector molecules produced by the colon tissues in the organ culture efficiently kill Escherichia coli in vitro. This approach, therefore, can be utilized to dissect the role of pathogen- and danger-associated molecular patterns and their cellular receptors in regulating intestinal innate immune responses and antimicrobial host defense responses.
Citrus tristeza virus (CTV) [genus Closterovirus; family Closteroviridae] is one of the most important, economically devastating viruses of citrus worldwide. On citrus trees grafted onto sour orange rootstock, typical CTV symptoms include dieback and defoliation, stunting, curling and chlorotic leaves, stem-pitting, and pinholes below the bud union on the inner face of the bark (Moreno et al. 2008). This single-stranded, positive-sense RNA virus is most efficiently transmitted by the brown citrus aphid (Toxoptera citricida), but it can also be transmitted by other aphid species and through grafting of infected plant material onto healthy plants (Moreno et al 2008; Herron et al. 2006). In Fall 2020, leaf material for virus testing was collected from 13 navel orange trees (Citrus × sinensis) grafted onto Poncirus trifoliata rootstocks (including 'Flying Dragon') located in a citrus research orchard in Tifton, GA. Trees ranged in age from 2 to 10 years, with the younger trees having been grafted from cuttings taken from the older trees. The oldest of these trees was derived from cuttings taken in 2009 from an orange tree growing locally in a residential yard in Tifton; this parent tree was more than 15 years old when these cuttings were obtained and was no longer available for sampling as of 2020. Symptoms or other visible signs of disease had not been noted on any of the tested trees, and trees were chosen for testing prior to the further dissemination of this plant material. The presence of CTV was verified via molecular and serological testing. CTV infection was initially confirmed in 8 of 13 tested samples using the ImmunoStrip® for CTV assay (Agdia® Inc., Elkhart, IN, cat no: ISK 78900/0025) according to the manufacturer's instructions. RNA was extracted from leaf material collected from the 13 sampled trees using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). Following cDNA synthesis, samples were tested for the presence of CTV by reverse-transcription PCR using primer pair AR18F (5'-ATGTCAGGCAGCTTGGGAAATT-3') and AR18R (5'-TTCGTGTCTAAGTCRCGCTAAACA-3') which produces a 511 bp amplicon (Roy et al., 2005). PCR reactions confirmed the presence of CTV, with the same eight samples that had previously tested positive via Immunostrip® producing PCR fragments of the expected size. Amplified products from two of these samples were then sequenced using Sanger sequencing (Retrogen Inc, San Diego, CA, USA) and subjected to BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for further identification. Sequence analysis revealed that the obtained partial sequences (MW540805) from the p18 gene of both isolates were 100% identical to one another and shared 100% identity to corresponding sequences from CTV strain N4 (MK779711.1). To the best of our knowledge, this is the first report of CTV infecting citrus plants in Georgia. CTV could pose an imminent threat to the emerging citrus industry in Georgia if it were to become established in commercial citrus plantings either via the dissemination of infected plant material or via vector transfer of the virus under field conditions. While the brown citrus aphid is not known to be widespread in Georgia at this time, other CTV vectors are prevalent including the cotton aphid (Aphis gossypii) and the black citrus aphid (T. aurantia). Georgia citrus growers and plant propagators should be aware of this virus and take appropriate control measures to prevent the spread of this viral diseas.
Phytopythium vexans (de Bary) Abad, de Cock, Bala, Robideau, A. M. Lodhi & Levesque is an important waterborne and soil-inhabiting oomycete pathogen causing root and crown rot of various plants including certain woody ornamentals, fruit, and forest trees. Early and accurate detection of Phytopythium in the nursery production system is critical, as this pathogen is quickly transported to neighboring healthy plants through the irrigation system. Conventional methods for the detection of this pathogen are tedious, frequently inconclusive, and costly. Hence, a specific, sensitive, and rapid molecular diagnostic method is required to overcome the limitations of traditional identification. In the current study, loop-mediated isothermal amplification (LAMP) for DNA amplification was developed for the identification of P. vexans. It was evaluated using real-time and colorimetric assays. Several sets of LAMP primers were designed and screened, but PVLSU2 was found to be specific to P. vexans as it did not amplify other closely related oomycetes, fungi, and bacteria. Moreover, the developed assays were sensitive enough to amplify DNA up to 102 fg per reaction. The real-time LAMP assay was more sensitive than traditional PCR and culture-based methods to detect infected plant samples. In addition, both LAMP assays detected as few as 100 zoospores suspended in 100 ml water. These LAMP assays are anticipated to save time in P. vexans detection by disease diagnostic laboratories and research institutions and enable early preparedness in the event of disease outbreaks.
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 1x105 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.
