Species within the Ilyonectria macrodidyma complex are known plant pathogens and several are implicated as the causal agents of black foot disease of grapevines. The seven species within the complex can be identified by DNA sequencing of the histone H3 gene. In this study, a PCR-RFLP method to identify the species was developed. In silico digestion of the 500 bp histone H3 amplimer using MnlI showed that it could identify Ilyonectria sp. 1, Ilyonectria sp. 2, I. alcacerensis and I. macrodidyma. Subsequent in silico digestion with Hinf1 identified I. estremocensis, I. novozelandica and I. torresensis. The PCR-RFLP was validated using a collection of 40 I. macrodidyma complex isolates that had been recovered from symptomatic grapevines. Ilyonectria macrodidyma, I. novozelandica and I. torresensis were detected in that collection. Intraspecific polymorphism was only detected in I. torresensis. This method provides a rapid procedure for identifying individual species of the I. macrodidyma species complex

Jaspers, Marlene V.

Jones, Elizabeth E.

Outram, Megan

Ridgway, Hayley J.

New Zealand Plant Protection

Lincoln University Research Archive

151Diseases of apples & grapesNew Zealand Plant Protection 67: 151-156 (2014) www.nzpps.orgM.A. Outram, E.E. Jones, M.V. Jaspers and H.J. RidgwayPlant Microbiology Group, Faculty of Agriculture and Life Sciences, Lincoln University,  PO Box 84, Lincoln 7647, New ZealandCorresponding author: Megan.Outram@lincoln.ac.nzAbstract Species within the Ilyonectria macrodidyma complex are known plant pathogens and several are implicated as the causal agents of black foot disease of grapevines. The seven species within the complex can be identified by DNA sequencing of the histone H3 gene. In this study, a PCR-RFLP method to identify the species was developed. In silico digestion of the 500 bp histone H3 amplimer using MnlI showed that it could identify Ilyonectria sp. 1, Ilyonectria sp. 2, I. alcacerensis and I. macrodidyma. Subsequent in silico digestion with Hinf1 identified I. estremocensis, I. novozelandica and I. torresensis. The PCR-RFLP was validated using a collection of 40 I. macrodidyma complex isolates that had been recovered from symptomatic grapevines. Ilyonectria macrodidyma, I. novozelandica and I. torresensis were detected in that collection. Intraspecific polymorphism was only detected in I. torresensis. This method provides a rapid procedure for identifying individual species of the I. macrodidyma species complex.Keywords Cylindrocapon, Ilyonectria macrodidyma, I. torresensis, I. novozelandica, Ilyonectria sp. 1, Ilyonectria sp. 2, I. alcacerensis, PCR-RFLP.Development of a PCR-RFLP method to distinguish species within the Ilyonectria macrodidyma complexINTRODUCTIONBlack foot disease of grapevine is a major disease associated with decline and death of vines, particularly in young vineyards and nurseries, in all major viticulture regions worldwide (Agusti-Brisach & Armengol 2013). This disease has been reported to have an increased incidence and severity over the past few years causing substantial economic losses (Oliveira et al. 2004; Halleen et al. 2006a). Several Cylindrocarpon-like species residing in the genera Campylocarpon and Ilyonectria have been identified as the causal agents of this disease. The predominant pathogens are Ilyonectria liriodendri and the I. macrodidyma complex, and two Campylocarpon species, C. fasciculare and C. pseudofasciculare (Cabral et al. 2012b). Although the relative importance, frequency and geographic distribution of these pathogens are still poorly understood, the I. liriodendri and I. macrodidyma complex are most commonly isolated from affected grapevines (Petit & Gubler 2005; Halleen et al. 2006a; Alaniz et al. 2007). In a New Zealand survey, Pathrose (2012) also found the I. radicicola complex was predominantly isolated from symptomatic grapevine material. Recent reclassification of Ilyonectria spp. has shown, however, that many of the earlier records actually represent some © 2014 New Zealand Plant Protection Society (Inc.) www.nzpps.org     Refer to http://www.nzpps.org/terms_of_use.html152Diseases of apples & grapesnewly described species (Cabral et al. 2012a, b). These include I. alcacerensis A. Cabral, Oliveira & Crous, I. estremocensis A. Cabral, Nascimento & Crous, I. novozelandica A. Cabral & Crous and I. torresensis A. Cabral, Rego & Crous, which were described from within the I. macrodidyma species complex (Cabral et al. 2012b).At present, sequence analysis is the only reliable method for the detection of the individual species of this complex. Sequence analysis is, however, expensive to carry out, especially when many isolates need to be analysed on a routine basis. Other methods include the use of morphological and cultural characteristics. Reis et al. (2013) found that conidial measurements allowed for clustering of isolates into seven distinct groups in accordance to the species groups as described by Cabral et al. (2012b), but cultural characteristics including mycelium coloration and growing margin revealed nine different groups of isolates. RAPD and ISSR markers have been successfully used to differentiate between the individual species, although low reproducibility of results means that their use is limited (Reis et al. 2013). This research aimed to develop a PCR-RFLP method to provide a rapid and sensitive method that could be routinely used to distinguish the individual species within the I. macrodidyma species complex recovered from grapevines.MATERIALS AND METHODSFungal isolatesThis study included 40 isolates of “C.” macrodidyma fungi, previously identified using morphological and molecular methods (Pathrose 2012). These isolates were obtained in a 2005 survey of symptomatic grapevine material of vineyards across seven regions (Auckland, Marlborough, Nelson, Gisborne, Central Otago, Waipara and Hawke’s Bay) in New Zealand and were maintained as agar slopes at 4°C (Bleach 2013). In the current study, these isolates were sequenced for part of the histone H3 gene (previously shown to be a very informative locus; Cabral et al. (2012b)) to identify the individual species within the I. macrodidyma complex. Sequencing reactions were performed by the Bio-Protection Research Centre, Lincoln University, Canterbury, New Zealand. The sequences obtained were then edited to remove any ambiguities using DNAMAN™ (LynnonBiosoft version 5.0) and compared to sequences present in the GenBank database (www.ncbi.nlm.nih.gov/Genbank/) using the nucleotide basic local alignment search tool (BLAST) algorithm (http://www.ncbi.nlm.nih.giv/BLAST/).DNA extraction and amplification For each isolate, genomic DNA was extracted using the PUREGENE genomic DNA isolation kit (PUREGENE®, GentraSystems, Minneapolis, USA) according to the manufacturer’s instructions. PCR amplification of the histone H3 gene was performed using the protocol described by Cabral et al. (2012b) using the CYLH3F and CYLH3R primers (Crous et al. 2004). PCR products were separated on a 1% agarose gel and stained by immersion in ethidium bromide (0.5 µg/ml) for 15 min and visualised under UV light. Molecular masses of products were estimated by comparison to a 1 kB Plus DNA ladder™ (Invitrogen).Design of PCR-RFLP analysis To rapidly identify the seven species belonging to the I. macrodidyma complex, a PCR-RFLP method was designed based on the nucleotide sequence of histone H3 gene of I. macrodidyma complex representatives (Table 1). These sequences were aligned by multiple species alignment using DNAMAN™ (LynnonBiosoft version 5.0). Areas of variability between the species were identified and at least 20 bp fragments were examined using the database of 180 frequently used enzymes available in DNAMAN™. Restriction digestion in silico was carried out and predicted restriction profiles were determined for each of the species. A list of readily available, inexpensive enzymes able to distinguish between the different species was compiled. Enzyme systems were then designed, beginning with an enzyme that was able to distinguish between three or more species in the initial digest. Several systems were developed in silico and the system that used the fewest number of enzymes was chosen.© 2014 New Zealand Plant Protection Society (Inc.) www.nzpps.org     Refer to http://www.nzpps.org/terms_of_use.html153Diseases of apples & grapesRestriction endonuclease digestionRestriction digestion in silico was carried out on representative sequences (Table 1) using a MnlI/HinfI system using DNAMAN™ (LynnonBiosoft version 5.0). Experimentally, amplimers of the histone H3 gene generated from the 40 “C.” macrodidyma isolates were digested with MnlI and HinfI (New England Biolabs). The reaction composition was as follows: (1) for MnlI, 5 U of enzyme was added to 14 µl of PCR product in 1× CutSmart™ Buffer (New England Biolabs, Inc.) in a total volume of 50 µl and (2) for HinfI, 10 U of enzyme was added to 14 µl of PCR product in 1× SuRE/Cut Buffer H (Roche, New Zealand) in a total volume of 50 µl. Digestions were carried out at 37°C for 16 h and stopped by heating to 65°C for 20 min as recommended by the manufacturer. For each species, 20 µl of the restriction digest product was mixed with 5 µl of loading dye (40% [w/v] sucrose, 0.25% bromophenol blue, 0.25% xylene cyanol). The first and last wells on the gel were loaded with 7 µl of 1 kB Plus DNA LadderTM (0.1 ng/µl) (Invitrogen, New Zealand) and 3 µl of loading dye. DNA fragments were separated by gel electrophoresis in a 10% polyacrylamide gel (acrylamide to bis ratio 19:1; Biorad) in 1× TBE buffer under a constant voltage of 270 V for 2 h 30 min. Gels were stained by immersion in ethidium bromide (0.5 µg/ml) for 15 min and visualised under UV light. Resultant DNA banding patterns were visually analysed to determine restriction profiles.RESULTSRestriction analysis of representative sequences in silicoThe in silico restriction patterns generated by MnlI are shown in Figure 1 and fragment sizes in Table 2. Isolates belonging to the species Ilyonectria sp. 1 produced banding pattern A (10 bands: range 9-192 bp), Ilyonectria sp. 2 produced banding pattern B (8 bands: range 9-201 bp), I. alcacerensis produced banding pattern C (9 bands: range 9-99 bp) and I. macrodidyma produced banding pattern E (8 bands: range 9-136 bp). The banding patterns produced from I. estremocensis (D) (8 bands: range 9-133), I. novozelandica (F) (7 bands: range 23-137) and I. torresensis (G) (8/9 bands: range 9-137) were unable to be differentiated from one another as Table 1 Representative DNA sequences of the histone H3 gene used for in silico PCR-RFLP analysis.SpeciesGenBank accession numbers  for histone H3 geneIlyonectria sp. 1 JF35610Ilyonectria sp. 2 JF735611I. alcacerensis JF735630I. novozelandica JF735633I. macrodidyma JF735647, JF735648I. torresensis JF735681, AM419087I. estremocensis JF735617Table 2 Restriction patterns of representative DNA sequences using restriction enzymes MnlI and HinfI applied to the histone H3 500 bp amplimer in silico.MnlI HinfISpeciesRFLPpattern Fragment size (bp)RFLP pattern Fragment size (bp)Ilyonectria sp. 1 A 192, 62, 54, 46, 37, 33, 24, 23, 20, 9   No digestionIlyonectria sp. 2 B 201, 70, 61, 40, 36, 36, 24, 23, 9   No digestion  No digestionI. alcacerensis C 99, 79, 68, 61, 56, 40, 31, 23, 9I. estremocensis D 133, 127, 75, 62, 40, 31, 23, 9 J 187, 94, 85, 70, 55, 9I. macrodidyma E 136, 130, 68, 61, 33, 23, 23, 16, 9   No digestionI. novozelandica F 137, 130, 70, 68, 39, 32, 23 H 351, 84, 50, 9I. torresensis G 137, 99, 68, 61, 40, 32, 31, 23, 9137, 130, 68, 61, 40, 32, 23, 9I 161, 127, 84, 63, 56, 9© 2014 New Zealand Plant Protection Society (Inc.) www.nzpps.org     Refer to http://www.nzpps.org/terms_of_use.html154Diseases of apples & grapesthe restriction fragments were ≤4 bp different in size or were reliant on the presence/absence of a 9 bp fragment. Digestion of I. estremocensis, I. novozelandica or I. torresensis using the restriction endonuclease HinfI produced distinctive banding patterns for these three species. Ilyonectria estremocensis produced banding pattern J (six bands: range 9-187 bp), I. novozelandica produced banding pattern H (four bands: range 9-351 bp) and I. torresensis produced banding pattern I (six bands: range 9-161 bp) (Figure 2 & Table 2).PCR using primers CYLH3F and CYLH3R yielded a single amplimer of approximately 500 bp for each isolate. The PCR-RFLP rapid identification system using restriction enzymes MnlI and HinfI was applied to the collection of 40 New Zealand isolates. It was found that the collection comprised three species I. macrodidyma (58%; n=23), I. novozelandica (22%; n=9) and I. torresensis (20%; n=8). Isolates of I. macrodidyma produced banding pattern E when digested with MnlI, while I. novozelandica and I. torresensis produced banding patterns F and G, respectively (Figure 3). Isolates belonging to I. torresensis produced the expected two variants in the banding pattern with MnlI digestion. Ilyonectria novozelandica and I. torresensis isolates were then digested with HinfI and produced banding patterns H and I (Figure 4).DISCUSSIONThe histone H3 gene has been shown to be useful in the distinction of the seven monophyletic species within the I. macrodidyma complex as described by Cabral et al. (2012b). In the present study a PCR-RFLP technique was developed using the 500 bp DNA fragment amplified from part of the histone H3 gene. PCR-RFLP analysis has previously been shown to be useful as a rapid method for taxonomic resolution of fungal plant pathogens, including closely Figure 1 In silico analysis of international representative sequences using the restriction enzyme MnlI. Lane 1: Ilyonectria sp. 1 (JF35610); Lane 2: Ilyonectria sp. 2 (JF735611); Lane 3: I. alcacerensis (JF735630); Lane 4: I. estremocensis (JF735617); Lanes 5-6: I. macrodidyma (JF735647, JF735648); Lane 7: I. novozelandica (JF735633); and Lanes 8-9: I. torresensis (JF735681, AM419087). The groups to which these species were allocated (A–G) are indicated at the bottom. Figure 2 In silico analysis of international representative sequences using the restriction enzyme HinfI. Lane 1: I. estremocensis (JF735617); Lane 2: I. novozelandica (JF735633); and Lanes 3-4: I. torresensis (JF735681, AM419087). The groups to which these species were allocated (H–J) are indicated at the bottom.© 2014 New Zealand Plant Protection Society (Inc.) www.nzpps.org     Refer to http://www.nzpps.org/terms_of_use.html155Diseases of apples & grapesFigure 3 Histone H3 PCR products digested with MnlI restriction enzyme visualised on a 10% polyacrylamide gel. Lanes L: 1 kB plus DNA ladder; Lanes 1-4: I. macrodidyma – Ack1c, Gis3d, Ack1a, Mar9c; Lanes 5-8: I. novozelandica – Ack2g, Co6c, Mar11f, Gis4a; and Lanes 9-12: I. torresensis – Ack2c, Hb2b, Wpa4a, Hb2c.Figure 4 Histone H3 PCR products digested with HinfI restriction enzyme visualised on a 10% polyacrylamide gel. Lanes L: 1 kB plus DNA ladder; Lanes 1-4: I. novozelandica – Ack2g, Co6c, Mar11f, Gis4a; and Lanes 5-8: I. torresensis – Ack2c, Hb2b, Wpa4, Hb2c.related grapevine pathogens species such as Botryosphaeria species (Alves et al. 2005). However, PCR-RFLP has never been applied to the I. macrodidyma species complex.The design of the PCR-RFLP procedure presented here involved analysis of 150 available restriction enzymes for the detection of variable regions in the histone H3 gene between the species within the I. macrodidyma species complex. The restriction enzyme, MnlI was chosen as it was able to distinguish the greatest number of species in a single digest (four species). It was necessary to also use the restriction enzyme HinfI as the banding patterns of I. estremocensis, I. novozelandica and I. torresensis following digestion with MnlI could not be differentiated from one another due to insufficient resolution of restriction fragments that were ≤4 bp different in molecular weight. These enzymes were also chosen as they are commonly available and inexpensive, meaning that this technique is cost effective, especially in comparison to DNA sequencing. This rapid and inexpensive tool will facilitate large scale studies on the population composition of these pathogen species in vineyards and other crop systems. As this technique involves sequence analysis it is reproducible and definitive for species identification and amenable to computer database analysis. However, this technique is unable to detect sequence differences at sites other than the MnlI and HinfI recognition sites resulting in its inability to detect other sequence variations.The PCR-RFLP procedure designed in this study was able to differentiate between these cryptic species using restriction analysis of the histone H3 gene. This gene was chosen as it has been shown by Cabral et al. (2012b) to have the highest resolving power (4% percent diversity or 20 bp difference in nucleotide sequence). Other genes including the β-tubulin (0.8% diversity) and the internal transcribed spacers on both sides of the 5.8S nuclear ribosomal RNA gene (ITS) (0% diversity) are ineffective at resolving these species. The MnlI/HinfI PCR-RFLP procedure presented here identified all seven of the I. macrodidyma complex species in silico and experimentally between isolates belonging to I. macrodidyma, I. torresensis and I. novozelandica. Intraspecific variability was detected in the © 2014 New Zealand Plant Protection Society (Inc.) www.nzpps.org     Refer to http://www.nzpps.org/terms_of_use.html156Diseases of apples & grapesrepresentative sequences of the international I. torresensis  isolates in silico and in the New Zealand I. torresensis isolates experimentally, following digestion with the restriction endonuclease, MnlI. Two distinctive patterns were produced for this species due to expected single nucleotide polymorphisms in the DNA sequence. However, despite this variation further digestion with HinfI produced a single distinctive pattern for this species allowing for definitive identification. The PCR-RFLP procedure presented here confirmed for the first time that three species belonging to the I. macrodidyma species complex are found in New Zealand. The most predominant species found in this collection of isolates was I. macrodidyma, with I. novozelandica and I. torresensis being present in approximately equal numbers. ACKNOWLEDGEMENTSThe authors would like to thank Lincoln University for funding this project and the Lincoln University Alumni Association for providing a postgraduate scholarship.REFERENCESAgusti-Brisach C, Armengol J 2013. Black-foot disease of grapevine: An update on taxonomy, epidemiology and management strategies. Phytopathologia Mediterranea 52(2): 245-261.Alaniz S, León M, Vicent A, García-Jiménez J, Abad-Campos P, Armengol J 2007. Characterisation of Cylindrocarpon species associated with black foot disease of grapevine in Spain. The American Phytopathological Society 91(9): 1187-1193.Alves A, Phillips AJL, Henriques I, Correia A 2005. Evaluation of amplified ribosomal DNA restriction analysis as a method for the identification of Botryosphaeria species. FEMS Microbiology Letters 245: 221-229.Bleach C 2013. Management of Cylindrocarpon black foot disease in New Zealand nurseries and vineyards. PhD thesis, Lincoln Univeristy, Christchurch, New Zealand.Cabral A, Groenewald JZ, Rego C, Oliveira H, Crous PW 2012a. Cylindrocarpon root rot: Multi-gene analysis reveals novels Ilyonectria radicicola species complex. Mycological Progress 11(3): 655-688.Cabral A, Rego C, Nascimento T, Oliveira H, Groenewald JZ, Crous PW 2012b. Multi-gene analysis and morphology reveal novel Ilyonectria species associated with black foot disease of grapevines. Fungal Biology 116(1): 62-80.Crous PW, Groenewald JZ, Risède J-M, Simoneau P, Hywel-Jones NL 2004. Calonectria species and their Cylindrocladium anamorphs: species with sphaeropedunculate vesicles. Studies in Mycology 50: 415–430.Halleen F, Fourie PH, Crous PW 2006a. A review of black foot disease of grapevine. Phytopathologia Mediterranea 45: S55-S67.Oliveira H, Rego MC, Nascimento T 2004. Decline of young grapevines caused by fungi. Acta Horticulture 652: 295-304.Pathrose B 2012. Characterising sub-species variation in New Zealand Cylindrocarpon species that cause black foot of grapevines. PhD thesis, Lincoln University, Christchurch, New Zealand.Petit E, Gubler WD 2005. Characterization of Cylindrocarpon species, the cause of black foot disease of grapevine in California. The American Phytopathological Society 89(10): 1051-1059.Reis P, Cabral A, Nascimento T, Oliveira H, Rego C 2013. Diversity of Ilyonectria species in young vineyard affected by black foot disease. Phytopathologia Mediterranea 52(2): 335-346.© 2014 New Zealand Plant Protection Society (Inc.) www.nzpps.org     Refer to http://www.nzpps.org/terms_of_use.html

Development of a PCR-RFLP method to distinguish species within the Ilyonectria macrodidyma complex

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