Rapid Detection of Colletotrichum siamense from Infected Tea Plants Using Filter-Disc DNA Extraction and Loop-Mediated Isothermal Amplification.

The pathogen Colletotrichum siamense causes tea anthracnose, resulting in economic losses to the Chinese tea industry. To effectively diagnose this pathogen in the field, we developed a loop-mediated isothermal amplification (LAMP) method using highly specific primers with a sensitivity of 1 pg/µl designed for amplifying the CAL gene, which was 10 times higher than that of conventional PCR. Additionally, to improve the method for obtaining DNA samples required for on-site diagnosis, we used the filter-disc DNA extraction method, which does not require special instruments and can be completed in a few minutes, and found that it effectively meets the requirements for the LAMP reaction. Finally, we combined LAMP with a filter-disc DNA extraction method (FDE-LAMP) to diagnose different degrees of disease in inoculated samples and 20 samples from the field. The results showed that the procedure had sufficient sensitivity for pathogen detection. Therefore, the FDE-LAMP procedure could greatly contribute to managing and preventing tea anthracnose in the field.

Synergistic effect of amino acid substitutions in CYP51B for prochloraz resistance in Fusarium fujikuroi.

Prochloraz has been used to control Fusarium fujikuroi, the causative pathogen of rice bakanae disease. Linkage analysis of FfCYP51 genes in the progenies obtained from crossing prochloraz moderately resistant and sensitive strains suggested that the FfCYP51B gene is involved in prochloraz resistance. Sequence comparison revealed that the prochloraz-resistant strain had an F511S or S312T/F511S substitution in FfCYP51B compared with the sensitive strains. The contribution of the S312T and F511S substitutions in FfCYP51B to prochloraz resistance was investigated by creating S/F-, T/F-, or T/S- types at 312/511 codons from the S/S-type, which is a natural moderately resistant strain, using a gene-editing technique. T/S exhibited the highest prochloraz resistance, followed by S/S-, T/F-, and S/F-types. These results indicated that the S312T and F511S substitutions in FfCYP51B had a synergistic effect on prochloraz resistance in F. fujikuroi.

A LAMP-Based Toolbox Developed for Detecting the Major Pathogens Affecting the Production and Quality of the Chinese Medicinal Crop Aconitum carmichaelii.

Aconitum carmichaelii Debeaux is a traditional Chinese medicinal herb that has been utilized for approximately 2,000 years. However, as cultivation has increased, there have been more reports of A. carmichaelii infections caused by four major pathogenic fungal species, Fusarium oxysporum, F. solani, Mucor circinelloides, and Sclerotium rolfsii, resulting in increased disease incidences and limited production and quality. To detect these infections, we developed a LAMP-based toolbox in this study. The cytochrome c oxidase subunit 1 (cox1) gene, translation elongation factor-1α (EF-1α), internal transcribed spacer (ITS) regions of rDNA, and alcohol dehydrogenase 1 (ADH1) gene, respectively, were used to design species-specific LAMP primer sets for F. oxysporum, F. solani, S. rolfsii, and M. circinelloides. The results showed that the LAMP-based toolbox was effective at detecting pathogens in soil and plant materials. We also used this toolbox to investigate pathogen infection in the main planting regions of A. carmichaelii. Before harvesting, F. oxysporum, M. circinelloides, and S. rolfsii were commonly found in the planting fields and in infected A. carmichaelii plants. Therefore, the toolbox we developed will be useful for tracking these infections, as well as for disease control in A. carmichaelii.

Correction: High-efficiency and high-fidelity ssDNA circularisation via the pairing of five 3'-terminal bases to assist LR-LAMP for the genotyping of single-nucleotide polymorphisms.

Correction for 'High-efficiency and high-fidelity ssDNA circularisation via the pairing of five 3'-terminal bases to assist LR-LAMP for the genotyping of single-nucleotide polymorphisms' by Taiwen Li et al., Analyst, 2022, https://doi.org/10.1039/d2an01042a.

High-efficiency and high-fidelity ssDNA circularisation via the pairing of five 3'-terminal bases to assist LR-LAMP for the genotyping of single-nucleotide polymorphisms.

The poor fidelity of T4 DNA ligase has always limited the simple detection of single-nucleotide polymorphisms (SNPs) and is only applicable to some special SNP types. This study developed a highly sensitive and specific detection method for SNPs based on high-fidelity single-stranded circularisation. It used T4 DNA ligase and rolling circle amplification (RCA) plus loop-mediated isothermal amplification (LAMP). Surprisingly, the cyclisation stage's efficiency greatly improved. The ligation fidelity was almost perfect via the unique pairing pattern between a long-paired base at the 5' terminus and only five bases at the 3' terminus on linear single-stranded DNA (l-DNA). Subsequently, LR-LAMP was performed and combined with the circularisation step for the simple detection of SNPs. The results showed that even 100 aM targets could be detected correctly and that a mutation rate of 0.1% or even 0.01% could be analysed via naked-eye visualisation or fluorescence detection, respectively. In addition, genomic DNA samples were used to evaluate the method, which indicated that it could effectively distinguish the SNPs of RPA190-T1145A in Phytophthora infestans. This strategy may play an important role in both circularisation of single-stranded DNA and detecting arbitrary SNPs.

Molecular Diagnosis of Thiophanate-Methyl-Resistant Strains of Fusarium fujikuroi in Japan.

Fusarium fujikuroi is the pathogen of rice bakanae disease and is subclassified into gibberellin and fumonisin groups (G and F groups). Thiophanate-methyl (TM), a benzimidazole fungicide, has been used extensively to control F. fujikuroi. Previous investigation showed that F-group strains are TM sensitive (TMS), whereas most G-group strains are TM resistant (TMR) in Japan. The minimum inhibitory concentration in TMS strains was 1 to 10 µg ml-1, whereas that in TMR strains was >100 µg ml-1. E198K and F200Y mutations in ß2-tubulin were detected in TMR strains. A loop-mediated isothermal amplification-fluorescent loop primer method was developed for diagnosis of these mutations and applied to 37 TMR strains and 56 TMS strains. The results indicated that 100% of TMR strains were identified as having either the E198K mutation (41%) or the F200Y mutation (59%), whereas none of the TMS strains tested showed either mutation. We found one remarkable TMR strain in the F group that had an F200Y mutation. These results suggest that E198K and F200Y mutations in ß2-tubulin contribute to TM resistance in F. fujikuroi.

Population Dynamics, Effective Soil Factors, and LAMP Detection Systems for Phytophthora Species Associated with Kiwifruit Diseases in China.

China has the largest area of kiwifruit production in the world. Pathogens associated with root diseases of kiwi trees have not been investigated extensively. In this research, three Phytophthora species, Phytophthora cactorum, Phytophthora cinnamomi, and Phytophthora lateralis, which are pathogenic to kiwi trees in the main planting areas of China, were studied. The population densities of these species in 128 soil samples from 32 kiwi orchards in 2017 and 2018 were measured using multiplex real-time quantitative PCR based on the ras-related protein gene Ypt1. P. cactorum was the most widely distributed of the three species in orchards of the Zhouzhi and Meixian prefectures. We used redundancy analysis to examine soil factors in the kiwi orchards to understand their effects on the population densities of the Phytophthora species. The redundancy analysis indicated that soil temperature and pH were significantly correlated with the abundance of P. cactorum and P. cinnamomi. In addition, two loop-mediated isothermal amplification detection systems for P. cactorum were developed based on the tigA gene. The color-change detection system proved to be accurate, sensitive, and faster than quantitative PCR. The results of this study, along with the loop-mediated isothermal amplification detection systems, will be of great use in the control of Phytophthora diseases for the production of kiwifruits in China.

Detection of the Genus Phytophthora and the Species Phytophthora nicotianae by LAMP with a QProbe.

Phytophthora is an oomycete genus with worldwide distribution, and many of its species cause destructive diseases. In Japan, Phytophthora species are listed as quarantine organisms with the exception of Phytophthora nicotianae. For effective quarantine control, we designed a Phytophthora genus-specific loop-mediated isothermal amplification (LAMP) primer set and a P. nicotianae species-specific quenching probe (QProbe) to establish a simultaneous LAMP-based detection method. We confirmed the specificity of the genus-specific primers, and all 161 taxa were detected. No other species in the closely related genera Pythium and Phytopythium gave positive results with the exception of two species, Phytopythium delawarense and Phytopythium fagopyri. These two species gave inconsistent results. We used annealing curve analysis with the QProbe to demonstrate that P. nicotianae could be distinguished from other species. DNA from inoculated and naturally infected plants was extracted using a time-saving extraction kit and subjected to the simultaneous detection method. We confirmed that all Phytophthora DNAs in the plant samples were detected, and P. nicotianae was specifically identified. This simultaneous detection method will make quarantine inspections faster and easier.

Genetic Differentiation Associated with Fumonisin and Gibberellin Production in Japanese Fusarium fujikuroi.

Fusarium fujikuroi is a pathogenic fungus that infects rice. It produces several important mycotoxins, such as fumonisins. Fumonisin production has been detected in strains of maize, strawberry, and wheat, whereas it has not been detected in strains from rice seedlings infested with bakanae disease in Japan. We investigated the genetic relationships, pathogenicity, and resistance to a fungicide, thiophanate-methyl (TM), in 51 fumonisin-producing strains and 44 nonproducing strains. Phylogenetic analyses based on amplified fragment length polymorphism (AFLP) markers and two specific genes (a combined sequence of translation elongation factor 1α [TEF1α] and RNA polymerase II second-largest subunit [RPB2]) indicated differential clustering between the fumonisin-producing and -nonproducing strains. One of the AFLP markers, EATMCAY107, was specifically present in the fumonisin-producing strains. A specific single nucleotide polymorphism (SNP) between the fumonisin-producing and nonproducing strains was also detected in RPB2, in addition to an SNP previously found in TEF1α. Gibberellin production was higher in the nonproducing than in the producing strains according to an in vitro assay, and the nonproducing strains had the strongest pathogenicity with regard to rice seedlings. TM resistance was closely correlated with the cluster of fumonisin-nonproducing strains. The results indicate that intraspecific evolution in Japanese F. fujikuroi is associated with fumonisin production and pathogenicity. Two subgroups of Japanese F. fujikuroi, designated G group and F group, were distinguished based on phylogenetic differences and the high production of gibberellin and fumonisin, respectively.IMPORTANCEFusarium fujikuroi is a pathogenic fungus that causes rice bakanae disease. Historically, this pathogen has been known as Fusarium moniliforme, along with many other species based on a broad species concept. Gibberellin, which is currently known as a plant hormone, is a virulence factor of F. fujikuroi Fumonisin is a carcinogenic mycotoxin posing a serious threat to food and feed safety. Although it has been confirmed that F. fujikuroi produces gibberellin and fumonisin, production varies among strains, and individual production has been obscured by the traditional appellation of F. moniliforme, difficulties in species identification, and variation in the assays used to determine the production of these secondary metabolites. In this study, we discovered two phylogenetic subgroups associated with fumonisin and gibberellin production in Japanese F. fujikuroi.

LAMP Detection of Four Plant-Pathogenic Oomycetes and Its Application in Lettuce Fields.

In Kagawa Prefecture, Japan, the pathogens Phytophthora pseudolactucae, Pythium irregulare, Pythium uncinulatum, and Pythium spinosum have caused huge losses in lettuce production. We used loop-mediated isothermal amplification (LAMP) to analyze soil and plants in lettuce fields for the presence of these four pathogens. To develop an effective on-site detection method, we contrasted the Plant-LAMP and Plant Culture-LAMP procedures for plant samples, and five soil DNA extraction methods for soil samples. Plant-LAMP and a Soil DNA Isolation kit were selected to analyze three fields for the pathogen species present, infected sites, and level of soil contamination. We found that the same wilting symptoms could be caused by Phytophthora or Pythium, or a mixture of species from both genera. Ph. pseudolactucae infects the pith of the lettuce in aboveground parts, whereas Pythium spp. mainly infect roots. Ph. pseudolactucae and Py. uncinulatum caused disease more frequently than the other two pathogens. Furthermore, not all of the pathogens existed in the soil near infected lettuce plants. Therefore, the LAMP method can be used to diagnose pathogenic oomycetes in the field, and will be useful in the development of control strategies in lettuce production.

Use of LAMP Detection to Identify Potential Contamination Sources of Plant-Pathogenic Pythium Species in Hydroponic Culture Systems of Tomato and Eustoma.

Hydroponic culture systems are subject to high risks of diseases caused by zoosporic plant pathogens. Control is generally difficult because of the rapid spread of zoospores in the nutrient solutions. In Japan, tomato and eustoma, which are cultivated using the D-tray and nutrient film techniques, respectively, are susceptible to diseases caused by Pythium aphanidermatum and P. irregulare. We used loop-mediated isothermal amplification to identify potential contamination sources of these two pathogens by monitoring their presence in the water supply wells, seedling terraces, nutrient solutions, diseased plants, and ground soils of a tomato greenhouse complex and a eustoma greenhouse complex. The results indicated that the pathogens may enter the culture systems from the soils around the greenhouses. Entry most likely occurs when seedlings are moved from the seedling terraces to the greenhouses, and sterilization of the hydroponic systems may not be sufficient. Therefore, monitoring pathogens in the culture systems and ground soils is very important for the management and prevention of these diseases.

Phytophthora niederhauserii sp. nov., a polyphagous species associated with ornamentals, fruit trees and native plants in 13 countries.

A non-papillate, heterothallic Phytophthora species first isolated in 2001 and subsequently from symptomatic roots, crowns and stems of 33 plant species in 25 unrelated botanical families from 13 countries is formally described here as a new species. Symptoms on various hosts included crown and stem rot, chlorosis, wilting, leaf blight, cankers and gumming. This species was isolated from Australia, Hungary, Israel, Italy, Japan, the Netherlands, Norway, South Africa, Spain, Taiwan, Turkey, the United Kingdom and United States in association with shrubs and herbaceous ornamentals grown mainly in greenhouses. The most prevalent hosts are English ivy (Hedera helix) and Cistus (Cistus salvifolius). The association of the species with acorn banksia (Banksia prionotes) plants in natural ecosystems in Australia, in affected vineyards (Vitis vinifera) in South Africa and almond (Prunus dulcis) trees in Spain and Turkey in addition to infection of shrubs and herbaceous ornamentals in a broad range of unrelated families are a sign of a wide ecological adaptation of the species and its potential threat to agricultural and natural ecosystems. The morphology of the persistent non-papillate ellipsoid sporangia, unique toruloid lobate hyphal swellings and amphigynous antheridia does not match any of the described species. Phylogenetic analysis based on sequences of the ITS rDNA, EF-1α, and ß-tub supported that this organism is a hitherto unknown species. It is closely related to species in ITS clade 7b with the most closely related species being P. sojae. The name Phytophthora niederhauserii has been used in previous studies without the formal description of the holotype. This name is validated in this manuscript with the formal description of Phytophthora niederhauserii Z.G. Abad et J.A. Abad, sp. nov. The name is coined to honor Dr John S. Niederhauser, a notable plant pathologist and the 1990 World Food Prize laureate.

A simple graphene oxide-based DNA purification strategy for plant pathogen detection.

BACKGROUND: The on-site molecular detection of plant pathogens is particularly important for the development of sustainable agriculture. Extracting DNA from plant tissues, microbes or coexisting environments is complex, labor-intensive and time-consuming. To facilitate this process, we propose a DNA purification strategy based on graphene oxide (GO). RESULTS: The excellent adsorption ability of GO was verified by visualizing changes in its microscopic surface and macroscopic mixture. To further optimize the DNA purification, we determined the optimal GO concentration and treatment time at 95 °C (2 mg mL-1 and 2 min, respectively). We confirmed that our strategy is effective on plant tissues and various microorganisms, and that the obtained DNA can be directly used for polymerase chain reaction amplification. Combining the proposed GO-based DNA purification method with the loop-mediated isothermal amplification method is superior, in terms of the required steps, time, cost and detection effect, to the cetyltrimethylammonium bromide method and a commercial kit for detecting plant pathogens. CONCLUSION: We present a feasible, rapid, simple and low-cost DNA purification method with high practical value for scientific applications in plant pathogen detection. This strategy can also provide important technical support for future research on plant-microbial microenvironments. © 2024 Society of Chemical Industry.

A species-specific method for detecting pathogenic Streptomyces species from soil and potato tubers in Argentina.

Potato common scab is caused by several soil-inhabiting pathogenic Streptomyces species. In the present study, a species-specific PCR method was used to detect Streptomyces species in potato tuber lesions and soils. Total genomic DNA from soil samples from six locations and tuber samples from four potato cultivars (Spunta, Shepody, Innovator and Russet Burbank) were assessed. Streptomyces scabies, Streptomyces acidiscabies, and Streptomyces turgidiscabies were detected in soybean, tobacco and potato soils and in all potato varieties except Russet Burbank. The phylogenetic analysis of the sequences obtained confirmed the identification. The method proposed proved to be time-saving and cost effective for the rapid detection of Streptomyces species. This is the first report of the detection of S. acidiscabies and S. turgidiscabies in soils and potato tubers from Argentina.

Population Genetic Analysis of Phytophthora colocasiae from Taro in Japan Using SSR Markers.

Phytophthora colocasiae is an important pathogen that causes great economic losses in taro production in tropical and subtropical regions, especially in Japan. Understanding the genetic variations in P. colocasiae populations and their transmission patterns in Japan is essential for effective disease control. Here, the genetic diversity of 358 P. colocasiae isolates, including 348 from Japan, 7 from China, and 3 from Indonesia, was assessed using 11 simple sequence repeat (SSR) primer pairs with high polymorphism. The phylogenetic tree of the SSR locus showed that the isolates from Japan could be divided into 14 groups, with group A being the dominant group. Among foreign isolates, only six from mainland China were similar to those from Japan and clustered in groups B and E. Analysis of molecular variance (AMOVA), principal components analysis (PCA), and cluster analysis (K = 3) results revealed a moderate level of genetic diversity, mainly within individuals. Populations showed high heterozygosity, a lack of regional differentiation, and frequent gene flow. Analysis of mating types and ploidy levels revealed that A2 and self-fertile (SF) A2 types and tetraploids were dominant across populations. Explanations and hypotheses for the results can provide more effective strategies for disease management of taro leaf blight.

Host Range and Loop-Mediated Isothermal Amplification Detection of Globisporangium sylvaticum from Guizhou, China.

Globisporangium, especially G. sylvaticum, causes devastating root rot, blight, and other diseases in various species of cash crops. To investigate the distribution and host range of G. sylvaticum in Guizhou, a suitable habitat for this pathogen, we collected 156 root-diseased samples, isolated the pathogens, and found that G. sylvaticum is widespread and has eleven host plants, including four novel hosts. Furthermore, to effectively identify G. sylvaticum, we developed a simple and dependable method based on loop-mediated isothermal amplification (LAMP), which used a primer set designed from the internal transcribed spacer sequences with high specificity and sensitivity of 1 pg/µL. Additionally, to perform field identification, we used the "Plant-LAMP" method with crude DNA extraction to detect the pathogen in 45 root samples from nine species of plants. Our results showed that this method could effectively detect G. sylvaticum in diseased roots. Therefore, our findings not only enrich existing research on the diversity of pathogenic Globisporangium in Guizhou but also present an efficient LAMP field detection method that could significantly contribute to plant disease management and prevention.

Phylogeography and population structure of the global, wide host-range hybrid pathogen Phytophthora × cambivora.

Invasive, exotic plant pathogens pose a major threat to native and agricultural ecosystems. Phytophthora × cambivora is an invasive, destructive pathogen of forest and fruit trees causing severe damage worldwide to chestnuts (Castanea), apricots, peaches, plums, almonds and cherries (Prunus), apples (Malus), oaks (Quercus), and beech (Fagus). It was one of the first damaging invasive Phytophthora species to be introduced to Europe and North America, although its origin is unknown. We determined its population genetic history in Europe, North and South America, Australia and East Asia (mainly Japan) using genotyping-by-sequencing. Populations in Europe and Australia appear clonal, those in North America are highly clonal yet show some degree of sexual reproduction, and those in East Asia are partially sexual. Two clonal lineages, each of opposite mating type, and a hybrid lineage derived from these two lineages, dominated the populations in Europe and were predominantly found on fagaceous forest hosts (Castanea, Quercus, Fagus). Isolates from fruit trees (Prunus and Malus) belonged to a separate lineage found in Australia, North America, Europe and East Asia, indicating the disease on fruit trees could be caused by a distinct lineage of P. × cambivora, which may potentially be a separate sister species and has likely been moved with live plants. The highest genetic diversity was found in Japan, suggesting that East Asia is the centre of origin of the pathogen. Further surveys in unsampled, temperate regions of East Asia are needed to more precisely identify the location and range of the centre of diversity.

A Novel Medium for Isolating Two Japanese Species in the Fusarium graminearum Species Complex and a Dipstick DNA Chromatography Assay for Species Identification and Trichothecene Typing.

Members of the Fusarium graminearum species complex (Fg complex) are the primary pathogens that cause Fusarium head blight in wheat and barley. Fg complex members grow poorly on Fusarium oxysporum-selective media, such as Komada and Fo-G2, that have also been used for the isolation of other Fusarium species. Therefore, Komada medium was modified as FG medium for the isolation of Fg complex members. However, the production of pentachloronitrobenzene that is the most effective component of FG medium is discontinued and new media is required for the selective isolation of Fg complex members. In addition, the rapid diagnosis of isolated fungi is useful for the disease control. Novel tools have been developed for isolating and characterizing Fg complex members. FG21, a semi-selective medium for isolating Fg complex members, was developed using potato dextrose agar. Furthermore, a dipstick DNA chromatography assay was developed both to identify Fusarium graminearum sensu stricto and Fusarium asiaticum in the Fg complex and their trichothecene mycotoxin types. The easier isolation and characterization of Fg complex members in Japan was attained by the combined use of FG21 medium and the dipstick DNA chromatography assay.

A Multiplex PCR for the Detection of Phytophthora nicotianae and P. cactorum, and a Survey of Their Occurrence in Strawberry Production Areas of Japan.

We aimed to simultaneously detect two pathogens causing strawberry diseases, Phytophthora nicotianae and P. cactorum, by multiplex polymerase chain reaction (PCR), and to survey their occurrence in the main strawberry production areas of Japan. Due to the need to combine different primer pairs for multiplex PCR and the low specificity of published specific primers for P. nicotianae and P. cactorum, new species-specific primers for P. nicotianae and P. cactorum were designed based on the internal transcribed spacer regions of ribosomal DNA and the ras-related protein gene Ypt1, respectively. Specificity of the designed primers was demonstrated using 68 isolates, including Phytophthora spp., Pythium spp., and other soilborne pathogens. Multiplex PCR discriminated between P. nicotianae and P. cactorum in DNA mixtures of mycelia of the two species. Moreover, both species were detected in artificially and naturally infested soils, indicating that these markers can be used in diagnosis of strawberry diseases. For investigation of the geographic distribution of the two pathogens in Japan, soil samples were collected in 89 strawberry fields from eight prefectures (Gifu, Saga, Nara, Tochigi, Chiba, Shizuoka, Yamanashi, and Hokkaido) of Japan. The method that was developed was successfully applied to survey P. nicotianae and P. cactorum, and distribution of the two pathogens in strawberry plantings in Japan was determined.

Multiplex LAMP Detection of the Genus Phytophthora and Four Phytophthora Species P. ramorum, P. lateralis, P. kernoviae, and P. nicotianae, with a Plant Internal Control.

Phytophthora species cause destructive plant diseases worldwide. All Phytophthora species, except for one, are listed as plant quarantine organisms in Japan. The exception, Phytophthora nicotianae is considered to be a domestic species. The injurious pests Phytophthora ramorum, Phytophthora lateralis, and Phytophthora kernoviae are invasive pathogens that cause tree mortality worldwide, mainly in the United States and the United Kingdom. To effectively control Phytophthora diseases, we established detection methods that utilize the loop-mediated isothermal amplification (LAMP) of the genus Phytophthora and the four species P. ramorum, P. lateralis, P. kernoviae, and P. nicotianae. LAMP primers for P. ramorum, P. lateralis, and P. kernoviae were newly designed in the present study. Our multiplex assay includes the detection of plant DNA as an internal control. When the optimum ratio between plant and pathogen primers was used in multiplex LAMP assays, 1 pg to 100 fg of pathogen DNA was detected with similar sensitivity to that in simplex LAMP assays. The detection of plant DNA in the absence of pathogens enables us to check for and avoid undesirable negative results caused by enzyme inactivation or the contamination of amplification inhibitors from plant tissues. The total time from sample collection to results is approximately 120| |min, and, thus, our multiplex LAMP assay may be used as an accurate and time-saving detection method for Phytophthora pathogens.