A loop-mediated isothermal amplification-based method for confirmation of Guignardia citricarpa in citrus black spot lesions

2013 ◽  
Vol 136 (2) ◽  
pp. 217-224 ◽  
Author(s):  
J. A. Tomlinson ◽  
S. Ostoja-Starzewska ◽  
K. Webb ◽  
J. Cole ◽  
A. Barnes ◽  
...  
Keyword(s):  
Black Spot ◽  
Isothermal Amplification ◽  
Guignardia Citricarpa ◽  
Loop Mediated Isothermal Amplification ◽  
Citrus Black Spot

scholarly journals A One-Day Sensitive Method to Detect and Distinguish Between the Citrus Black Spot Pathogen Guignardia citricarpa and the Endophyte Guignardia mangiferae

Plant Disease ◽  
2006 ◽  
Vol 90 (1) ◽  
pp. 97-101 ◽  
Author(s):  
L. Meyer ◽  
G. M. Sanders ◽  
R. Jacobs ◽  
L. Korsten
Keyword(s):  
South African ◽  
Sequence Data ◽  
Black Spot ◽  
The United States ◽  
Guignardia Citricarpa ◽  
Competitive Edge ◽  
Citrus Black Spot ◽  
Species Specific ◽  
Time Required ◽  
Slow Growing

If South African citrus exporters wish to retain their competitive edge in the European market and access new markets such as the United States of America, it is of quarantine importance to distinguish between the citrus black spot pathogen, Guignardia citricarpa, and the harmless endophyte, G. mangiferae. The endophyte is not a sanitary or phytosanitary concern. This paper describes the design of species-specific primers that are able to detect and distinguish between these two Guignardia species. Application of the primer set CITRIC1 and CAMEL2 in conjunction with the ITS4 primer yielded polymerase chain reaction (PCR) amplicons of approximately 580 bp and 430 bp for G. citricarpa and G. mangiferae, respectively. Results obtained with these primers are in accordance with sequence data, and repeated tests verified accuracy and sensitivity. A BLAST search revealed no matches other than G. citricarpa and G. mangiferae, and no positive PCR results were obtained with Colletotrichum gloeosporioides, which is the most common contaminant in black spot lesions. We are, therefore, able to distinguish G. citricarpa and G. mangiferae unequivocally using a PCR-based method. This method was further improved to directly isolate DNA from fruit lesions by means of the DNeasy Plant Mini Kit (Qiagen). This eliminates the prior need for culturing the slow-growing organism, thereby shortening the time required to one day to test for and verify the presence or absence of the pathogenic G. citricarpa in export consignments.

scholarly journals Speciesspecific PCR primers for Guignardia citricarpa and Guignardia mangiferae

2006 ◽  
Vol 59 ◽  
pp. 141-145 ◽  
Author(s):  
K.R. Everett ◽  
J. Rees-George
Keyword(s):  
New Zealand ◽  
Venturia Inaequalis ◽  
Black Spot ◽  
Pathogenic Fungi ◽  
Pcr Primers ◽  
Colletotrichum Acutatum ◽  
Botryosphaeria Dothidea ◽  
Guignardia Citricarpa ◽  
Citrus Black Spot ◽  
Polymerase Chain

The plant pathogen Guignardia citricarpa causes citrus black spot and is not considered to be present in New Zealand Speciesspecific polymerase chain reaction (PCR) primers were designed to identify G citricarpa and G mangiferae a closely related saprotroph that is present in New Zealand These PCR primers were tested against a range of other saprotrophic and pathogenic fungi viz Botrytis cinerea Botryosphaeria dothidea B parva Cladosporium sp Colletotrichum acutatum C gloeosporioides Cryptosporiopsis sp Epicoccum sp Nigrospora sp Penicillium sp Pestalotia sp Phialophora sp Phlyctema sp Phoma sp Phomopsis sp Stemphylium sp and Venturia inaequalis The primers JRGGc were specific to G citricarpa and JRGGm to G mangiferae A 226 bp product was amplified from G mangiferae DNA using JRGGm primers and a 501 bp product was amplified from G citricarpa DNA using JRGGc primers These primers thus distinguished G citricarpa from G mangiferae and can be used to rapidly identify incursions by citrus black spot

scholarly journals Rapid Detection ofAlternariaSpecies Involved in Pear Black Spot Using Loop-Mediated Isothermal Amplification

Plant Disease ◽  
2019 ◽  
Vol 103 (12) ◽  
pp. 3002-3008 ◽  
Author(s):  
Xue Yang ◽  
Yong-Jie Qi ◽  
Mohamed N. Al-Attala ◽  
Zheng-Hui Gao ◽  
Xing-Kai Yi ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Black Spot ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Cyt B ◽  
Spot Disease ◽  
Loop Mediated Isothermal Amplification ◽  
Yellowish Green ◽  
Alternaria Species ◽  
Cyt B Gene

Alternaria species are the most important fungal pathogens that attack various crops as well as fruit trees such as pear and cause black spot disease. Here, a loop-mediated isothermal amplification (LAMP) assay is developed for the detection of Alternaria species. A. alternata cytochrome b (cyt-b) gene was used to design two pairs of primers and amplified a 229-bp segment of Aacyt-b gene. The results showed that LAMP assay is faster and simpler than polymerase chain reaction (PCR). LAMP assay is highly sensitive method for the detection of about 1 pg of genomic DNA of A. alternata by using optimized concentration of MgCl2(4 mM) in final LAMP reaction. In contrast, the limit of detection was 1 ng of target DNA via conventional PCR. Among the genomic DNA of 46 fungal species, only the tubes containing DNA of Alternaria spp. except A. porri, A. solani, and A. infectoria changed color from orange to yellowish green with SYBR Green I including the main pathogens of pear black spot. The yellowish green color was indicative of DNA amplification. Moreover, LAMP assay was used for testing infected tissues among 22 healthy and diseased pear tissues; the orange color changed to yellowish green for infected tissues only. Altogether, we conclude that cyt-b gene can be used for the detection of Alternaria spp. via LAMP assay, which is involved in pear black spot disease.

scholarly journals Application of Azoxystrobin for Control of Benomyl-Resistant Guignardia citricarpa on ‘Valencia’ Oranges in South Africa

Plant Disease ◽  
2003 ◽  
Vol 87 (7) ◽  
pp. 784-788 ◽  
Author(s):  
G. C. Schutte ◽  
R. I. Mansfield ◽  
H. Smith ◽  
K. V. Beeton
Keyword(s):  
South Africa ◽  
Disease Control ◽  
Untreated Control ◽  
Black Spot ◽  
Mineral Oil ◽  
Guignardia Citricarpa ◽  
Citrus Black Spot ◽  
Oil In Water ◽  
Small Plot

Azoxystrobin was evaluated in replicated small-plot trials from 1995 to 1999 for control of citrus black spot (CBS) on ‘Valencia’ oranges caused by Guignardia citricarpa. Applications of different rates of tank mixes of azoxystrobin and mancozeb during the susceptible period from October to January were compared with an untreated control as well as the standard four applications of mancozeb with or without mineral oil (1.20 g a.i./liter + 0.5% [vol/vol]/liter and 1.60 g a.i./liter of water, respectively). Two applications of azoxystrobin in tank mixtures with mancozeb and mineral oil (0.5% [vol/vol]/liter) in mid-November and mid-January at rates of 0.10, 0.15, and 0.20 g a.i./liter controlled CBS by more than 98 to 99%, 99 to 100% and 95 to 98%, respectively. Concomitantly, where mineral oil was not added to the fungicide mixture, azoxystrobin and mancozeb resulted only in 73 to 95%, 74 to 93% and 92.2 to 92.3% CBS control, respectively. Tank mixtures of benomyl, mancozeb, and mineral oil reduced CBS by only 29%, which could be attributed to the presence of benomyl-resistant pathogen isolates in the experimental orchard. Azoxystrobin applied at rates of 0.05, 0.075, and 0.10 g a.i./liter in tank mixtures with mancozeb (1.2 g a.i./liter) and mineral oil (0.5% [vol/vol]/liter of water) or Agral 90 (0.5% [vol/vol]/liter of water) were equally effective, reducing CBS by more than 99%. When mineral oil was compared to different adjuvants in tank mixtures with azoxystrobin and mancozeb, only mineral oil resulted in 100% clean exportable fruit. There was no difference between Sunspray 6E and Bac oil when mixed with azoxystrobin and mancozeb on the degree of disease control. Furthermore, the concentration of mineral oil in water can be lowered from 0.5% (vol/vol)/liter of water to 0.3% (vol/vol)/liter of water without a loss in efficacy against CBS. It is therefore, recommended that azoxystrobin (0.075 g a.i./liter) must be applied in tank mixtures with mancozeb (1.2 g a.i./liter) and mineral oil, which can be applied at either 0.5% (vol/vol)/liter of water or 0.3% (vol/vol)/liter of water.

scholarly journals High molecular diversity of the fungus Guignardia citricarpa and Guignardia mangiferae and new primers for the diagnosis of the citrus black spot

2009 ◽  
Vol 52 (5) ◽  
pp. 1063-1073 ◽  
Author(s):  
Danyelle Stringari ◽  
Chirlei Glienke ◽  
Daniel de Christo ◽  
Walter Maccheroni Jr. ◽  
João Lucio de Azevedo
Keyword(s):  
Rapd Markers ◽  
Geographical Origin ◽  
Black Spot ◽  
Polymerase Chain Reaction Analysis ◽  
Causal Agent ◽  
Specific Primers ◽  
Guignardia Citricarpa ◽  
Citrus Black Spot ◽  
Polymerase Chain ◽  
Sequence Characterized Amplified Regions

RAPD markers were used to investigate the distribution of genetic variability among a group of Guignardia citricarpa, G. mangiferae, and Phyllosticta spinarum isolates obtained from several hosts in Brazil, Argentina, Mexico, Costa Rica, Thailand, Japan, United States and South Africa. Pathogenic isolates G. citricarpa Kiely (anamorph form P. citricarpa McAlp Van Der Aa) are the etiological agent of the Citrus Black Spot (CBS), a disease that affects several citric plants and causes substantial injuries to the appearance of their fruits, thus preventing their export. Several previous studies have demonstrated the existence of an endophytic species with high morphological similarity to the causal agent of CBS that could remain latent in the same hosts. Consequently, the identification of the plants and fruits free from the causal agent of the disease is severely hampered. The RAPD analysis showed a clear discrimination among the pathogenic isolates of G. citricarpa and endophytic isolates (G. mangiferae and P. spinarum). In addition, a Principal Coordinate Analysis (PCO) based on a matrix of genetic similarity estimated by the RAPD markers showed four clusters, irrespective of their host or geographical origin. An Analysis of Molecular Variance (AMOVA) indicated that 62.8% of the genetic variation was found between the populations (G. citricarpa, G. mangiferae, P. spinarum and Phyllosticta sp.). Substantial variation was found in the populations (37.2%). Exclusive RAPD markers of isolates of G. citricarpa were cloned, sequenced and used to obtain SCARS (Sequence Characterized Amplified Regions), which allowed the development of new specific primers for the identification of G. citricarpa PCR (Polymerase Chain Reaction) analysis using a pair of primers specific to pathogenic isolates corroborating the groupings obtained by the RAPD markers, underscoring its efficiency in the identification of the causal agent of CBS.

scholarly journals Rapid and Specific Detection of the Poplar Black Spot Disease Caused by Marssonina brunnea Using Loop-Mediated Isothermal Amplification Assay

Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 253
Author(s):  
Qin Xiong ◽  
Linlin Zhang ◽  
Xinyue Zheng ◽  
Yulin Qian ◽  
Yaxin Zhang ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Fungal Endophytes ◽  
Black Spot ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Sensitive Detection ◽  
Spot Disease ◽  
Loop Mediated Isothermal Amplification ◽  
Marssonina Brunnea ◽  
Black Spot Disease

Marssonina brunnea is the main pathogen that causes poplar black spot disease, which leads to the decrease of the photosynthetic efficiency and significantly affects the production and quality of timber. Currently, no in-field diagnostic exists for M. brunnea. Here, we described a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of M. brunnea. A set of six oligonucleotide primers was designed to recognize eight distinct sequences of the internal transcribed spacer (ITS) region of M. brunnea. The LAMP assay was optimized by the combination of high specificity, sensitivity, and rapidity for the detection of less than 10 pg/μL of target genomic DNA in 60 min per reaction at 65 °C, whereas with PCR, there was no amplification of DNA with concentration less than 1 ng/μL. Among the genomic DNA of 20 fungalisolates, only the samples containing the genomic DNA of M. brunnea changed from violet to sky blue (visible to the naked eye) by using hydroxynaphthol blue (HNB) dye. No DNA was amplified from the eight other fungus species, including two other Marssonina pathogens, three other foliar fungi pathogens of poplar, and three common foliar fungal endophytes of poplar. Moreover, the detection rates of M. brunnea from artificially and naturally infected poplar leaves were 10/16 (62.5%) and 6/16 (37.5%) using PCR, respectively, while the positive-sample ratios were both 16/16 (100%) using the LAMP assay. Overall, the ITS LAMP assay established here can be a better alternative to PCR-based techniques for the specific and sensitive detection of M. brunnea in poplar endemic areas with resource-limited settings.

Biofertilizer for control of Guignardia citricarpa, the causal agent of citrus black spot

2006 ◽  
Vol 25 (6) ◽  
pp. 569-573 ◽  
Author(s):  
K.C. Kupper ◽  
W. Bettiol ◽  
A. de Goes ◽  
P.S. de Souza ◽  
J.A.M. Bellotte
Keyword(s):  
Black Spot ◽  
Causal Agent ◽  
Guignardia Citricarpa ◽  
Citrus Black Spot
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