LAMP assay for distinguishing Fusarium oxysporum and Fusarium commune in lotus (Nelumbo nucifera) rhizomes

Plant Disease ◽  
2021 ◽  
Author(s):  
Sheng Deng ◽  
Xin Ma ◽  
Yifan Chen ◽  
Hui Feng ◽  
Dongmei Zhou ◽  
...  
Keyword(s):  
Fusarium Oxysporum ◽  
Fusarium Species ◽  
Lamp Assay ◽  
Rapid Identification ◽  
Nelumbo Nucifera ◽  
Disease Spread ◽  
Molecular Diagnostic ◽  
Primer Sets ◽  
Rot Disease ◽  
Rhizome Rot

The yields of edible rhizome from the cultivation of the perennial hydrophyte lotus (Nelumbo nucifera) can be severely reduced by rhizome rot disease caused by Fusarium species. There is a lack of rapid field-applicable methods for detection of these pathogens on lotus plants displaying symptoms of rhizome-rot. Fusarium commune (91%) and Fusarium oxysporum (9%) were identified at different frequencies from lotus samples showing symptoms of rhizome-rot. As these two species can cause different severity of disease and their morphology is very similar, molecular-diagnostic based methods to detect these two species were developed. Based on the comparison of the mitochondrial genome of the two species, three specific DNA loci targets were found. The designed primer sets for conventional PCR, qPCR and loop-mediated isothermal amplification (LAMP) precisely distinguished the above two species when isolated from lotus and other plants. The LAMP detection limits were 10 pg/μl and 1 pg/μl of total DNA for F. commune and F. oxysporum, respectively. We also carried out field-mimicked experiments on lotus seedlings and rhizomes (including inoculated samples and field diseased samples), and the results indicated that the LAMP primer sets and the supporting portable methods are suitable for the rapid diagnosis of the lotus disease in the field. The LAMP-based detection method will aid in the rapid identification of whether F. oxysporum or F. commune are infecting lotus plants with symptoms of rhizome-rot, and can facilitate efficient pesticide use and prevent the disease spread through vegetative propagation of Fusarium-infected lotus rhizomes.

scholarly journals Intensitas dan Luas Serangan Beberapa Isolat Fusarium oxysporum f.sp. zingiberi pada Jahe Gajah

2017 ◽  
Vol 21 (1) ◽  
pp. 16
Author(s):  
Hermawati Cahyaningrum ◽  
Nur Prihatiningsih ◽  
Soedarmono Soedarmono
Keyword(s):  
Fusarium Oxysporum ◽  
Sterile Soil ◽  
Soil Medium ◽  
Disease Symptoms ◽  
Ginger Rhizome ◽  
Incubation Periods ◽  
Rot Disease ◽  
Screen House ◽  
Rhizome Rot ◽  
Growth Ability

Ginger is one of the spices and medicinal commodities which is cultivated in Indonesia. One of the obstacles encountered in the cultivation of ginger is the rhizome rot disease which is mainly caused by Fusarium oxysporum Schlecht f.sp. zingiberi Trujillo. This study is aimed to know the growth ability and virulence level of the isolates on ginger rhizome and plants. The research was conducted in the laboratory and in the screen house by using Complete Random Design consisted of 10 treatments and 4 replications. The parameters observed were growth ability of F. oxysporum f.sp. zingiberi, rhizome rot disease symptoms, incubation period, extensive decay and weight difference of the rhizomes. The results showed that F. oxysporum f.sp. zingiberi which was stored for 4 years in sterile soil medium was still capable to cause damage to the rhizome and plants. Incubation periods of rhizome decay and plant symptoms were from 3 to 11.5 and 55.5 to 68.5 days, respectively. The most virulent isolate was MSO1 with extensive decay of rhizome and the wilting intensity were 108.95 mm2 dan 33.88%, respectively. IntisariJahe merupakan salah satu komoditas rempah dan obat yang banyak dibudidayakan di Indonesia. Salah satu kendala yang dihadapi dalam budidaya jahe adalah adanya gangguan penyakit busuk rimpang yang disebabkan (terutama) oleh Fusarium oxysporum Schlecht f.sp. zingiberi Trujillo. Penelitian bertujuan untuk menguji daya tumbuh dan virulensi isolat F. oxysporum f.sp. zingiberi pada rimpang dan tanaman jahe gajah. Penelitian dilakukan di laboratorium dan di rumah kasa menggunakan Rancangan Acak Kelompok Lengkap (RAKL) yang masing-masing terdiri dari 10 perlakuan dan 4 ulangan. Parameter yang diamati meliputi daya tumbuh F. oxysporum f.sp. zingiberi, gejala penyakit busuk rimpang, masa inkubasi, luas pembusukan dan selisih bobot basah rimpang. Hasil penelitian menunjukkan bahwa F. oxysporum f.sp. zingiberi yang telah di simpan 4 tahun dalam medium tanah steril mampu menyebabkan kerusakan pada rimpang dan tanaman jahe. Masa inkubasi gejala busuk pada rimpang serta gejala pada tanaman masing- masing berkisar antara 3–11,5 serta 55,5–68,5 hari. Isolat yang paling virulen adalah MSO1 dengan nilai luas pembusukan pada rimpang dan intensitas penyakit masing-masing sebesar 108,95 mm2 dan 33,88%.

scholarly journals Development of a loop-mediated isothermal amplification (LAMP) assay for the identification of the invasive wood borer Aromia bungii (Coleoptera: Cerambycidae) from frass

3 Biotech ◽  
2021 ◽  
Vol 11 (2) ◽  
Author(s):  
Domenico Rizzo ◽  
Nicola Luchi ◽  
Daniele Da Lio ◽  
Linda Bartolini ◽  
Francesco Nugnes ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Isothermal Amplification ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Loop Mediated Isothermal Amplification ◽  
Larval Stages ◽  
Longhorn Beetle ◽  
Rapid Monitoring ◽  
Major Pest ◽  
Wood Borer

AbstractThe red-necked longhorn beetle Aromia bungii (Faldermann, 1835) (Coleoptera: Cerambycidae) is native to east Asia, where it is a major pest of cultivated and ornamental species of the genus Prunus. Morphological or molecular discrimination of adults or larval specimens is required to identify this invasive wood borer. However, recovering larval stages of the pest from trunks and branches causes extensive damage to plants and is timewasting. An alternative approach consists in applying non-invasive molecular diagnostic tools to biological traces (i.e., fecal pellets, frass). In this way, infestations in host plants can be detected without destructive methods. This paper presents a protocol based on both real-time and visual loop-mediated isothermal amplification (LAMP), using DNA of A. bungii extracted from fecal particles in larval frass. Laboratory validations demonstrated the robustness of the protocols adopted and their reliability was confirmed performing an inter-lab blind panel. The LAMP assay and the qPCR SYBR Green method using the F3/B3 LAMP external primers were equally sensitive, and both were more sensitive than the conventional PCR (sensitivity > 103 to the same starting matrix). The visual LAMP protocol, due to the relatively easy performance of the method, could be a useful tool to apply in rapid monitoring of A. bungii and in the management of its outbreaks.

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

2021 ◽  
Vol 22 (2) ◽  
pp. 822
Author(s):  
Owen Hudson ◽  
Sumyya Waliullah ◽  
James C. Fulton ◽  
Pingsheng Ji ◽  
Nicholas S. Dufault ◽  
...  
Keyword(s):  
South Carolina ◽  
Fusarium Oxysporum ◽  
Fusarium Wilt ◽  
Conventional Polymerase Chain Reaction ◽  
Detection Methods ◽  
Limiting Factors ◽  
Pcr Marker ◽  
Primer Sets ◽  
Race Differentiation ◽  
Race 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.

scholarly journals Ice nucleation activity identified in some phytopathogenic Fusarium species

2005 ◽  
Vol 77 (2) ◽  
pp. 83-92 ◽  
Author(s):  
C. Richard ◽  
J.-G. Martin ◽  
S. Pouleur
Keyword(s):  
Fusarium Oxysporum ◽  
Fusarium Species ◽  
Freezing Temperature ◽  
Ice Nucleation ◽  
Free Living ◽  
Factors Affecting ◽  
Pure Cultures ◽  
Nucleation Activity ◽  
Ice Nucleation Activity ◽  
Positive Controls

In order to know which species of Fusarium are ice nucleating and to determine the factors affecting their pathogenicity, ice nucleation activity (INA) was examined in Fusarium oxysporum, F. sporotrichioides, and F. tricinctum. Positive controls (lna+) used were F. acuminatum and F. avenaceum. The test for fungal INA was done with a simple and rapid tube nucleation assay. Twelve out of the 42 F. oxysporum isolates, and 8 out of 14 F. tricinctum isolates were lna+. No INA was detected in F sporotrichioides. In this test the threshold freezing temperature tended to increase with culture age, reaching a peak of -1°C in a few samples, which is as high as the warmest INA reported for bacteria, and higher than the INA detected in pure cultures of free-living fungi, lichen fungi, lichen algae and cyanobacteria. This is the first report of INA for F oxysporum.

scholarly journals Fusarium Consortium Populations Associated with Asparagus Crop in Spain and Their Role on Field Decline Syndrome

2020 ◽  
Vol 6 (4) ◽  
pp. 336
Author(s):  
Alexandri María Brizuela ◽  
Eduardo De la Lastra ◽  
José Ignacio Marín-Guirao ◽  
Laura Gálvez ◽  
Miguel de Cara-García ◽  
...  
Keyword(s):  
Fusarium Oxysporum ◽  
Fusarium Species ◽  
Management Strategies ◽  
Statistical Correlation ◽  
Inoculum Density ◽  
Average Temperature ◽  
Species Complexes ◽  
Main Production ◽  
Production Areas ◽  
Prevalent Species

Asparagus Decline Syndrome (ADS) is one of the main phytosanitary problems of asparagus crop worldwide. Diseased plants and soil samples from 41 fields from three main production areas of Spain were surveyed. Eight Fusarium species belonging to seven species complexes were identified in soils: F. oxysporum, F. proliferatum, F. redolens, F. solanisensu stricto, F. equiseti, F. culmorum, F. compactum and F. acuminatum. Fusarium oxysporum was the most prevalent species. Statistical correlation (R2 = 88%) was established between F. oxysporum inoculum density and the average temperature of the warmest month. A relationship was also established between three crop factors (average temperature, crop age and F. oxysporum inoculum density) and field disease indices. Significant differences were observed between the distribution of F. oxysporum propagules in white and green asparagus fields. Thirteen Fusarium species belonging to seven species complexes were identified from roots of diseased plants, being F. oxysporum the most prevalent. F. proliferatum, F. oxysporum and F. redolens showed pathogenicity to asparagus and were the main species associated to ADS. Fusarium oxysporum was the species with the highest genetic diversity displaying 14 sequence-based haplotypes with no geographic differentiation. This work contributes to understanding the Fusarium complex associated to ADS for developing accurate integrated disease management strategies.

scholarly journals Detection and Control of Fusarium oxysporum from Soft Rot in Dendrobium officinale by Loop-Mediated Isothermal Amplification Assays

Biology ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1136
Author(s):  
Caiyun Xiao ◽  
Rongyu Li
Keyword(s):  
Fusarium Oxysporum ◽  
Integrated Management ◽  
Elongation Factor ◽  
Lamp Assay ◽  
Soft Rot ◽  
Isothermal Amplification ◽  
Dendrobium Officinale ◽  
Control Effect ◽  
Loop Mediated Isothermal Amplification ◽  
And Control

Soft rot causing Fusarium oxysporum is one of the most destructive diseases of Dendrobium officinale Kimura et Migo in China that reduces D. officinale yield and quality. A key challenge for an integrated management strategy for this disease is the rapid and accurate detection of F. oxysporum on D. officinale. Therefore, a new loop-mediated isothermal amplification (LAMP) assay was developed for this purpose. In this study, the primers were selected and designed using the translation elongation factor-1α (TEF-1α) gene region as the target DNA sequence in order to screen the best system of reaction of LAMP to detect F. oxysporum through optimizing different conditions of the LAMP reaction, including time, temperature, concentrations of MgSO4, and concentrations of inner and outer primers. The optimized system was able to efficiently amplify the target gene at 62 °C for 60 min with 1.2 μM internal primers, 0.4 μM external primers, 7 mM Mg2+, and 5 fg/µL minimum detection concentration of DNA for F. oxysporum. The amplified products could be detected with the naked eye after completion of the reaction with SYBR green I. We were better able to control the effect of soft rot in D. officinale using fungicides following a positive test result. Additionally, the control effect of synergism combinations against soft rot was higher than 75%. Thus, LAMP assays could detect F. oxysporum in infected tissues of D. officinale and soils in field, allowing for early diagnosis of the disease.

scholarly journals Standard and molecular methods in the diagnostics of infections caused by equine herpesviruses 1 and 4

2018 ◽  
Vol 72 (2) ◽  
pp. 68-79
Author(s):  
Nenad Milic ◽  
Andrea Radalj ◽  
Jakov Nisavic
Keyword(s):  
Screening Method ◽  
Rapid Identification ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Equine Herpesvirus ◽  
Viral Kinetics ◽  
Herpesvirus Type ◽  
Equine Herpesvirus Type 1 ◽  
Herpesvirus 4 ◽  
Different Strains

Background. Equine herpesvirus type 1 (EHV-1) is responsible for respiratory disease in young animals, abortion in pregnant mares and neurological disease, whilst equine herpesvirus 4 (EHV-4) is mainly the causative agent of respiratory disorders and rarely causes abortion. These viruses are considered as one of the most clinically and economically important pathogens of horses and can be detected in a range of tissues. Scope and Approach. Serological methods are used to detect the presence and titre of specific antibodies to EHV-1 and EHV-4 in the sera of examined horses and are useful in epizootiological studies. Commercially available ELISA kits are able to differentiate specific EHV-1 and EHV-4 antibodies. EHV-1 and EHV-4 can both be isolated using susceptible cells such as primary horse cell cultures and other non-equine cells with visible cytopathic effect. Since standard diagnostic methods can be time consuming and arduous, the scope of many studies has been to develop and confirm the sensitivity and specificity of molecular diagnostic methods. Key Findings and Conclusions. Polymerase chain reaction (PCR) has proved to be a good screening method for the presence of latent infections of horses caused by these viruses, also making possible the rapid identification and differentiation of EHV-1 and- EHV-4 in the examined samples. Real-time PCR is a sensitive, specific and quantitative method that enables the determination of viral kinetics in infected horses. Genome sequencing can be used to discover mutations in the genomes of EHV-1 and EHV-4 as well as to track the spread of their different strains globally.

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