Multiple Mutations and Overexpression in the CYP51A and B Genes Lead to Decreased Sensitivity of Venturia effusa to Tebuconazole.

Multiple demethylation-inhibiting (DMI) fungicides are used to control pecan scab, caused by Venturia effusa. To compare the efficacy of various DMI fungicides on V. effusa, field trials were conducted at multiple locations applying fungicides to individual pecan terminals. In vitro assays were conducted to test the sensitivity of V. effusa isolates from multiple locations to various concentrations of tebuconazole. Both studies confirmed high levels of resistance to tebuconazole. To investigate the mechanism of resistance, two copies of the CYP51 gene, CYP51A and CYP51B, of resistant and sensitive isolates were sequenced and scanned for mutations. In the CYP51A gene, mutation at codon 444 (G444D), and in the CYP51B gene, mutations at codon 357 (G357H) and 177 (I77T/I77L) were found in resistant isolates. Expression analysis of CYP51A and CYP51B revealed enhanced expression in the resistant isolates compared to the sensitive isolates. There were 3.0- and 1.9-fold increases in gene expression in the resistant isolates compared to the sensitive isolates for the CYP51A and CYP51B genes, respectively. Therefore, two potential mechanisms-multiple point mutations and gene over expression in the CYP51 gene of V. effusa isolates-were revealed as likely reasons for the observed resistance in isolates of V. effusa to tebuconazole.

Development of a CAPS Marker and a LAMP Assay for Rapid Detection of Xylella fastidiosa Subsp. multiplex and Differentiation from X. fastidiosa Subsp. fastidiosa on Blueberry.

Bacterial leaf scorch (BLS), caused by Xylella fastidiosa (Xf), is a prevalent disease of blueberries in the southeastern United States. Initially, this disease was reported to be caused by X. fastidiosa subsp. multiplex (Xfm). However, a recent survey revealed the presence of another subspecies, X. fastidiosa subsp. fastidiosa (Xff), within naturally infected blueberry plantings in Georgia. Since knowledge regarding the origins of isolates causing Xf outbreaks can impact management recommendations, a routine method for identifying the pathogen at the subspecies level can be beneficial. Several detection strategies are available to identify Xf infection at the subspecies level. However, none of these have been developed for the routine and rapid differentiation of the blueberry-infecting Xf subspecies. Here, we developed two separate straightforward and rapid detection techniques, a cleaved amplified polymorphic sequence (CAPS) marker, and a loop-mediated isothermal amplification (LAMP) assay, targeting the RNA polymerase sigma-70 factor (rpoD) gene sequence of Xfm to discriminate between the two Xf subspecies infecting blueberry. With the CAPS marker, specific detection of Xfm isolates was possible from pure cultures, inoculated greenhouse-grown plant samples, and field infected blueberry samples by restriction digestion of the rpoD gene PCR product (amplified with primers RST31 and RST33) using the BtsI enzyme. The LAMP assay allowed for specific real-time amplification of a 204-bp portion of the XfmrpoD gene from both pure bacterial cultures and infected plant material using the Genie® III system, a result further affirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. These detection strategies have the potential to greatly aid existing diagnostic methods for determining the distribution and prevalence of these Xf subspecies causing bacterial leaf scorch (BLS) in blueberries in the southeastern United States.

Fusarium oxysporum f. sp. niveum Molecular Diagnostics Past, Present and Future.

Watermelon is an important commercial crop in the Southeastern United States and around the world. However, production is significantly limited by biotic factors including fusarium wilt caused by the hemibiotrophic fungus Fusarium oxysporum forma specialis niveum (Fon). Unfortunately, this disease has increased significantly in its presence over the last several decades as races have emerged which can overcome the available commercial resistance. Management strategies include rotation, improved crop resistance, and chemical control, but early and accurate diagnostics are required for appropriate management. Accurate diagnostics require molecular and genomic strategies due to the near identical genomic sequences of the various races. Bioassays exist for evaluating both the pathogenicity and virulence of an isolate but are limited by the time and resources required. Molecular strategies are still imperfect but greatly reduce the time to complete the diagnosis. This article presents the current state of the research surrounding races, both how races have been detected and diagnosed in the past and future prospects for improving the system of differentiation. Additionally, the available Fon genomes were analyzed using a strategy previously described in separate formae speciales avirulence gene association studies in Fusarium oxysporum races.

Marker Development for Differentiation of Fusarium Oxysporum f. sp. Niveum Race 3 from Races 1 and 2.

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.

Characterization of Resistance to Major Tropical Root-Knot Nematodes (Meloidogyne spp.) in Solanum sisymbriifolium.

Root-knot nematodes (Meloidogyne spp.) are important contributors to yield reduction in tomato. Though resistant cultivars to common species (Meloidogyne arenaria, M. incognita, and M. javanica) are available, they are not effective against other major species of root-knot nematodes. Cultivars or lines of Solanum sisymbriifolium were examined to assess the presence and level of resistance to five major species: M. arenaria race 1, M. incognita race 3, M. haplanaria, M. javanica, and M. enterolobii. Differences in S. sisymbriifolium response to the nematode infection were apparent when susceptibility or resistance was classified by the egg counts per gram fresh weight of root and the multiplication rate of the nematodes. The cultivar Diamond was highly susceptible, Quattro and White Star were susceptible, while Sis Syn II was resistant to M. arenaria. Quattro, White Star, and Sis Syn II exhibited a moderate to high level of resistance to M. incognita but the nematode increased 2.5-fold from the initial population of the M. incognita on Diamond. All S. sisymbriifolium cultivars were highly resistant to both M. haplanaria and M. enterolobii, while highly susceptible to M. javanica. A microplot study under field conditions using Sis Syn II confirmed that M. arenaria, M. incognita, and M. haplanaria were not pathogenic on the plant. Likewise, an examination on cross-sections of galled root tissues confirmed the susceptibility and resistance of S. sisymbriifolium lines to Meloidogyne spp. Using S. sisymbriifolium as a resistant rootstock or a new source of resistance may result in the development of nonchemical and sustainable management strategies to protect the tomato crop.

First Report of Hop stunt viroid Infecting Citrus Trees in Georgia, USA.

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.

Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cucurbit Leaf Crumple Virus.

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.

Conferring virus resistance in tomato by independent RNA silencing of three tomato homologs of Arabidopsis TOM1.

The TOM1/TOM3 genes from Arabidopsis are involved in the replication of tobamoviruses. Tomato homologs of these genes, LeTH1, LeTH2 and LeTH3, are known. In this study, we examined transgenic tomato lines where inverted repeats of either LeTH1, LeTH2 or LeTH3 were introduced by Agrobacterium. Endogenous mRNA expression for each gene was detected in non-transgenic control plants, whereas a very low level of each of the three genes was found in the corresponding line. Small interfering RNA was detected in the transgenic lines. Each silenced line showed similar levels of tobamovirus resistance, indicating that each gene is similarly involved in virus replication.

Estimating water levels caused by a tropical storm along the Bangladesh coast: A numerical approach.

In this study, water levels resulting from the dynamic interaction of tide and surge are estimated by solving a 2-D vertically integrated shallow water equations numerically. To solve the equations on the specific 2-D grid, the explicit Leapfrog scheme is implemented, adopting a staggered Arakawa C-grid. The domain's complex land-sea interface is approximated through the stair-step method in order to employ the finite difference technique. To incorporate the complexity of the domain with a considerably high accuracy and to reduce computational cost, one-way nested grid models are embraced. The Meghna River freshwater discharge is incorporated into the innermost child model. A stable tidal regime over the region of interest is generated by applying the four vital tidal constituents, namely M2 (principal lunar semidiurnal), S2 (principal solar semidiurnal), O1 (principal lunar diurnal) and K1 (luni-solar diurnal) in the southern open boundary of the outermost model. This previously effectuated tidal regime is used as the initial state of the sea in getting total water levels due to the dynamic interaction of tide and surge. Numerical experiments are performed with the storm AILA that hit the coast of Bangladesh on May 25, 2009. The simulated results are found to closely match observed and reported data.

Loop-mediated isothermal amplification (LAMP) assay for specific and rapid detection of Dickeya fangzhongdai targeting a unique genomic region.

Dickeya fangzhongdai, a bacterial pathogen of taro (Colocasia esculenta), onion (Allium sp.), and several species in the orchid family (Orchidaceae) causes soft rot and bleeding canker diseases. No field-deployable diagnostic tool is available for specific detection of this pathogen in different plant tissues. Therefore, we developed a field-deployable loop-mediated isothermal amplification (LAMP) assay using a unique genomic region, present exclusively in D. fangzhongdai. Multiple genomes of D. fangzhongdai, and other species of Dickeya, Pectobacterium and unrelated genera were used for comparative genomic analyses to identify an exclusive and conserved target sequence from the major facilitator superfamily (MFS) transporter gene region. This gene region had broad detection capability for D. fangzhongdai and thus was used to design primers for endpoint PCR and LAMP assays. In-silico validation showed high specificity with D. fangzhongdai genome sequences available in the NCBI GenBank genome database as well as the in-house sequenced genome. The specificity of the LAMP assay was determined with 96 strains that included all Dickeya species and Pectobacterium species as well as other closely related genera and 5 hosts; no false positives or false negatives were detected. The detection limit of the assay was determined by performing four sensitivity assays with tenfold serially diluted purified genomic DNA of D. fangzhongdai with and without the presence of crude host extract (taro, orchid, and onion). The detection limit for all sensitivity assays was 100 fg (18-20 genome copies) with no negative interference by host crude extracts. The assays were performed by five independent operators (blind test) and on three instruments (Rotor-Gene, thermocycler and dry bath); the assay results were concordant. The assay consistently detected the target pathogen from artificially inoculated and naturally infected host samples. The developed assay is highly specific for D. fangzhongdai and has applications in routine diagnostics, phytosanitary and seed certification programs, and epidemiological studies.

A Core35S Promoter of Cauliflower Mosaic Virus Drives More Efficient Replication of Turnip Crinkle Virus.

The 35S promoter with a duplicated enhancer (frequently referred to as 2X35S) is a strong dicotyledonous plant-specific promoter commonly used in generating transgenic plants to enable high-level expression of genes of interest. It is also used to drive the initiation of RNA virus replication from viral cDNA, with the consensus understanding that high levels of viral RNA production powered by 2X35S permit a more efficient initiation of virus replication. Here, we showed that the exact opposite is true. We found that, compared to the Core35S promoter, the 2X35S promoter-driven initiation of turnip crinkle virus (TCV) infection was delayed by at least 24 h. We first compared three versions of 35S promoter, namely 2X35S, 1X35S, and Core35S, for their ability to power the expression of a non-replicating green fluorescent protein (GFP) gene, and confirmed that 2X35S and Core35S correlated with the highest and lowest GFP expression, respectively. However, when inserted upstream of TCV cDNA, 2X35S-driven replication was not detected until 72 h post-inoculation (72 hpi) in inoculated leaves. By contrast, Core35S-driven replication was detected earlier at 48 hpi. A similar delay was also observed in systemically infected leaves (six versus four days post-inoculation). Combining our results, we hypothesized that the stronger 2X35S promoter might enable a higher accumulation of a TCV protein that became a repressor of TCV replication at higher cellular concentration. Extending from these results, we propose that the Core35S (or mini35S) promoter is likely a better choice for generating infectious cDNA clones of TCV.

Molecular Characterization of Laboratory Mutants of Fusarium oxysporum f. sp. niveum Resistant to Prothioconazole, a Demethylation Inhibitor (DMI) Fungicide.

Fusarium oxysporum f. sp. niveum (FON) is the causal agent of Fusarium wilt in watermelon, an international growth-limiting pathogen of watermelon cultivation. A single demethylation inhibitor (DMI) fungicide, prothioconazole, is registered to control this pathogen, so the risk of resistance arising in the field is high. To determine and predict the mechanism by which FON could develop resistance to prothioconazole, FON isolates were mutagenized using UV irradiation and subsequent fungicide exposure to create artificially resistant mutants. Isolates were then put into three groups based on the EC50 values: sensitive, intermediately resistant, and highly resistant. The mean EC50 values were 4.98 µg/mL for the sensitive, 31.77 µg/mL for the intermediately resistant, and 108.33 µg/mL for the highly resistant isolates. Isolates were then sequenced and analyzed for differences in both the coding and promoter regions. Two mutations were found that conferred amino acid changes in the target gene, CYP51A, in both intermediately and highly resistant mutants. An expression analysis for the gene CYP51A also showed a significant increase in the expression of the highly resistant mutants compared to the sensitive controls. In this study, we were able to identify two potential mechanisms of resistance to the DMI fungicide prothioconazole in FON isolates: gene overexpression and multiple point mutations. This research should expedite growers' and researchers' ability to detect and manage fungicide-resistant phytopathogens.

Sensitivity of Aspergillus flavus Isolates from Peanut Seeds in Georgia to Azoxystrobin, a Quinone outside Inhibitor (QoI) Fungicide.

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.

Lump, lump-stripe, and breather wave solutions to the (2 + 1)-dimensional Sawada-Kotera equation in fluid mechanics.

The present study investigates the lump, one-stripe, lump-stripe, and breather wave solutions to the (2+1)-dimensional Sawada-Kotera equation using the Hirota bilinear method. For lump and lump-stripe solutions, a quadratic polynomial function, and a quadratic polynomial function in conjunction with an exponential term are assumed for the unknown function f giving the solution to the mentioned equation, respectively. On the other hand, only an exponential function is considered for one-stripe solutions. Besides, the homoclinic test approach is adopted for constructing breather wave solutions. The propagations of the attained lump, lump-stripe, and breather wave solutions are shown through some graphical illustrations. The graphical outputs demonstrate that the lump wave moves along the straight line y = - x and exponentially decreases away from the origin of the spatial domain. On the other hand, lump-kink solutions illustrate the fission and fusion interaction behaviors upon the selection of the free parameters. Fission and fusion processes show that the stripe soliton splits into a stripe soliton and a lump soliton, and then the lump soliton merges into one stripe soliton. In addition, the achieved breather waves illustrate the periodic behaviors in the xy-plane. The outcomes of the study can be useful for a better understanding of the physical nature of long waves in shallow water under gravity.

Solving protoplanetary structure equations using Adomian decomposition method.

In this paper, the distribution of thermodynamic variables in the protoplanets formed by gravitational instability in the mass range 0.3 - 10 M J ( 1 M J = 1 Jupiter mass = 1.8986 × 10 30 gm) is investigated in their initial state by solving the structure equations via the Adomian decomposition method. Concerning the heat transfer in the protoplanets, the mode of convection is taken into account. The outcomes indicate that there is a reasonably good agreement between the Adomian semi-analytical solution containing only first 8 terms and the numerical results.

Phylogenetic and phenotypic characterization of Fusarium oxysporum f. sp. niveum isolates from Florida-grown watermelon.

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.

Genome-informed loop-mediated isothermal amplification assay for specific detection of Pectobacterium parmentieri in infected potato tissues and soil.

Pectobacterium parmentieri (formerly Pectobacterium wasabiae), which causes soft rot disease in potatoes, is a newly established species of pectinolytic bacteria within the family Pectobacteriaceae. Despite serious damage caused to the potato industry worldwide, no field-deployable diagnostic tests are available to detect the pathogen in plant samples. In this study, we aimed to develop a reliable, rapid, field-deployable loop-mediated isothermal amplification (LAMP) assay for the specific detection of P. parmentieri. Specific LAMP primers targeting the petF1 gene region, found in P. parmentieri but no other Pectobacterium spp., were designed and validated in silico and in vitro using extensive inclusivity (15 strains of P. parmentieri) and exclusivity (94 strains including all other species in the genus Pectobacterium and host DNA) panels. No false positives or negatives were detected when the assay was tested directly with bacterial colonies, and with infected plant and soil samples. Sensitivity (analytical) assays using serially diluted bacterial cell lysate and purified genomic DNA established the detection limit at 10 CFU/mL and 100 fg (18-20 genome copies), respectively, even in the presence of host crude DNA. Consistent results obtained by multiple users/operators and field tests suggest the assay's applicability to routine diagnostics, seed certification programs, biosecurity, and epidemiological studies.

Turfgrass Disease Diagnosis: Past, Present, and Future.

Turfgrass is a multibillion-dollar industry severely affected by plant pathogens including fungi, bacteria, viruses, and nematodes. Many of the diseases in turfgrass have similar signs and symptoms, making it difficult to diagnose the specific problem pathogen. Incorrect diagnosis leads to the delay of treatment and excessive use of chemicals. To effectively control these diseases, it is important to have rapid and accurate detection systems in the early stages of infection that harbor relatively low pathogen populations. There are many methods for diagnosing pathogens on turfgrass. Traditional methods include symptoms, morphology, and microscopy identification. These have been followed by nucleic acid detection and onsite detection techniques. Many of these methods allow for rapid diagnosis, some even within the field without much expertise. There are several methods that have great potential, such as high-throughput sequencing and remote sensing. Utilization of these techniques for disease diagnosis allows for faster and accurate disease diagnosis and a reduction in damage and cost of control. Understanding of each of these techniques can allow researchers to select which method is best suited for their pathogen of interest. The objective of this article is to provide an overview of the turfgrass diagnostics efforts used and highlight prospects for disease detection.

Rapid detection of pecan root-knot nematode, Meloidogyne partityla, in laboratory and field conditions using loop-mediated isothermal amplification.

Meloidogyne partityla is the dominant root-knot nematode (RKN) species parasitizing pecan in Georgia. This species is known to cause a reduction in root growth and a decline in the yields of mature pecan trees. Rapid and accurate diagnosis of this RKN is required to control this nematode disease and reduce losses in pecan production. In this study, a loop-mediated isothermal amplification (LAMP) method was developed for simple, rapid, and on-site detection of M. partityla in infested plant roots and validated to detect the nematode in laboratory and field conditions. Specific primers were designed based on the sequence distinction of the internal transcribed spacer (ITS)-18S/5.8S ribosomal RNA gene between M. partityla and other Meloidogyne spp. The LAMP detection technique could detect the presence of M. partityla genomic DNA at a concentration as low as 1 pg, and no cross reactivity was found with DNA from other major RKN species such as M. javanica, M. incognita and M. arenaria, and M. hapla. We also conducted a traditional morphology-based diagnostic assay and conventional polymerase chain reaction (PCR) assay to determine which of these techniques was less time consuming, more sensitive, and convenient to use in the field. The LAMP assay provided more rapid results, amplifying the target nematode species in less than 60 min at 70°C, with results 100 times more sensitive than conventional PCR (~2-3 hrs). Morphology-based, traditional diagnosis was highly time-consuming (2 days) and more laborious than conventional PCR and LAMP assays. These features greatly simplified the operating procedure and made the assay a powerful tool for rapid, on-site detection of pecan RKN, M. partityla. The developed LAMP assay will facilitate accurate pecan nematode diagnosis in the field and contribute to the management of the pathogen.

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification.

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.