Identification of <Emphasis Type="Italic">Ganoderma</Emphasis>, the causal agent of basal stem rot disease in oil palm using a molecular method

From comparison of the alignments of the internally transcribed spacers (ITS) of ribosomal DNA from Ganoderma associated with oil palm basal stem rot (BSR) and other Ganoderma species, two specific primer pairs were selected to provide a specific DNA amplification of pathogenic Ganoderma in oil palm. Each primer pair produced a single PCR product of about 450 bp (for primer pair IT1–IT2) and 334 bp (for primer pair IT1–IT3) when oil palm Ganoderma DNA was used. No PCR amplification product was observed when other Ganoderma species DNA was used in PCR amplification with these primer pairs. Three specific restriction enzyme sites were identified in the ITS and intergenic spacer (IGS1) regions. The restriction enzymes MluI, SacI and HinfI were used to digest the ITS-PCR product and restriction enzymes TfiI, ScaI and HincII were used to digest the IGS1-PCR product. Of the three restriction enzymes used in each rDNA region, MluI specifically digested the ITS regions, and TfiI specifically digested the IGS1 region of oil palm Ganoderma. Analysis of the published ITS nucleotide sequences of 31 Ganoderma species showed that the MluI restriction site was not present in other Ganoderma species. The use of both specific primers and restriction enzyme analysis can be applied as a standard protocol to identify pathogenic Ganoderma in oil palm. In this study, the use of specific primers and PCR-RFLP analyses of the rDNA gave consistent results for the characterisation of pathogenic Ganoderma, and indicated that Ganoderma strains associated with BSR disease in oil palms belong to a single species.     

An insight into spore dispersal of <Emphasis Type="Italic">Ganoderma boninense</Emphasis> on oil palm

The disease of oil palm caused by Ganoderma boninense, although universally referred to as Ganoderma basal stem rot, occurs in three very distinct phases, with basal stem rot only part of the disease cycle. G. boninense also causes a seedling disease and an upper stem rot. An understanding of spore dispersal provides an insight into where spores of G. boninense have a role in the infection process. This role will be discussed in relation to each of these three infection phases. This understanding is a critical component of developing a successful disease control strategy.     

Stem rots of oil palm caused by <Emphasis Type="Italic">Ganoderma boninense</Emphasis>: Pathogen biology and epidemiology

Oil palm (Elaeis guineensis Jacq.) has been grown in Papua New Guinea since the early 1960s. The most important disease of oil palm in PNG is a stem rot of the palm base. This is the same disease that constitutes a major threat to sustainable oil palm production in SE Asia. Investigations into the causal pathogen have revealed that the stem rots in PNG are caused predominantly by the basidiomycete Ganoderma boninense, with a minor pathogen identified as G. tornatum G. tornatum was found to have a broad host range whereas G. boninense appears to be restricted to palms. The population structure of G. boninense was investigated using inter-fertility studies between isolates collected from basal stem rots on oil palm. Although the G. boninense field populations are predominantly comprised of distinct individuals, a number of isolates were found that share single mating alleles. This indicates that out-crossing had occurred over several generations in the resident or wild population of G. boninense prior to colonization of oil palm. No direct hereditary relationship between isolates on neighbouring diseased palms was found, although an indirect link between isolates causing upper stem rot and basal stem rot was detected.     

Constancy of RAPD primer amplification strength among distantly related taxa of flowering plants

A survey of 480 10-mer primers for RAPD markers revealed general consistency in primer amplification strength among the flowering plant generaDatisca, Helianthus andYucca. Six characteristics of primer base sequences were analyzed: total (G+C) content; the amounts of G, A, C, and T taken separately; and the (G+C) content in the last four bases of the 3′ end. Of these, total (G+C) content showed the most value in predicting primer amplification strength. Since the consistency of amplification strength is true only globally, there are still many primers showing high variation in amplification strength among genera, most probably due to DNA sequence differences, but perhaps also resulting from experimental artifact. Nevertheless, we suggest that this survey be used as a rough guide for prioritizing primer deployment in RAPD studies involving plants in the hope of improving efficiency during the search for adequate levels of polymorphism, with the understanding that taxon-specific differences in primer amplification strength are bound to occur.     

Molecular and biochemical studies of virus infections in two filamentous brown algae

The filamentous marine brown algae Ectocarpus siliculosus and Feldmannia simplex are infected by host specific DNA-viruses. Under Percoll isolation, Ectocarpus siliculosus-virus (EsV)-particles maintained their infective potential.The EsV has a circular genome of dsDNA with a size of 320 kb. A restriction map has been established. The gene of a major coat protein (gp1) was detected in a genomic library and partly sequenced. Using gpl- sequences for polymerase chain reaction (PCR) amplification analysis, EsV specific sequences could be detected in various symptom-free, clonal cultures of Ectocarpus. The PCR was also used to follow the passage of the virus genome during the meiosis of hosts. A monospecific antibody against recombinant gpl was used for immunostaining and infection experiments.The Feldmannia simplex-virus (FlexV-1) has a circular genome with a size of 220 kb and a 43% G+C content. FsV-DNA contains methylated bases. 5-methylcytosine (5 mC) makes up 12% of the total cytosines.     

PCR-based DNA Markers for Identifying Hybrids within Phytophthora alni

Two pairs of oligonucleotide primers were designed for the polymerase chain reaction (PCR)‐based detection and differential identification of naturally occurring interspecific hybrid types (subspecies) of Phytophthora alni, all of which cause collar rot of alder trees. Primer pairs were derived from randomly amplified polymorphic DNA (RAPD) fragments that were unique to various subspecies of this alder pathogen. The primer pair set, SAP1/SAP2 (SAP), was derived from a 0.93‐kb RAPD fragment amplified from P. alni ssp. alni. The primer pair set, SWAP1/SWAP2 (SWAP), was derived from a 1.13‐kb fragment amplified from P. alni ssp. uniformis. Patterns of SAP and SWAP amplification enabled distinction among the three subspecies. No PCR products were amplified from isolates of 31 other Phytophthora spp. examined, including P. cambivora and P. fragariae, the suspected progenitors of P. alni. The SAP and SWAP primer sets were able to detect a minimum of 10 pg of DNA from pure cultures or DNA extracted from as few as 10 zoospores. Pathogen DNA could also be amplified directly from bark lesions of artificially inoculated and naturally infected common alders and from lesions developed on common cherry‐laurel leaves used in baiting the pathogen from infested soil. Direct detection of pathogen DNA from alder tissue using SAP and SWAP primer sets should prove useful in developing measures for effective quarantine and management of P. alni.     

Rapid amplification of genomic DNA ends bynla III partial digestion and polynucleotide tailing

We report a simple and efficient method, which combines restriction endonuclease digestion and deoxynucleotide tailing, for cloning unknown genomic sequences adjacent to a known sequence. Total genomic DNA is partially digested with the frequent-cutting restriction enzymeNla III. A homo-oligomeric cytosine tail is added by terminal transferase. The tailed DNA fragments are used as the template for cloning flanking regions from all sequences of interest. A first round PCR amplification is performed with a gene-specific primer and the selective (modified polyguanine) anchor primer complementary to the cytosine tail and theNla III recognition site, with a universal amplification primer sequence at its 5′ end. This is followed by another PCR amplification with a nested gene-specific primer and the universal amplification primer. Finally, the amplified products are fractionated, cloned, and sequenced. Using this method, we cloned the upstream region of a salt-induced gene based upon a partial cDNA clone (RSC5-U) obtained from sunflower (Helianthus annuus L.).     

Production of Pectic Enzymes by Fusarium oxysporum f. sp. cepae and its Involvement in Onion Bulb Rot

Fusarium oxysporum f. sp. cepae produced an exo-polygalacturonase (exo-PG) and endopectin-trans-eliminase (endo-PTE) in a mineral medium supplemented with a restricted supply of either D-galacturonic acid or onion cell walls. These enzymes were also extracted from infected onion tissue, but only endo-PTE caused tissue maceration and cell death. The patterns of host tissue colonization and pectic enzyme production were followed during bulb rot development. Stem plates were invaded within two weeks of inoculation. The pathogen then remained confined to the stem plates for several weeks or months, before spreading to the outer fleshy scales to initiate a basal rot. In most cases the inner leaf sheaths containing the lateral bud remained healthy. Exo-PG activity m stem plate tissue was greatest at two weeks after inoculation, then it declined. Endo-PTE was not detected in newly invaded stem plate tissue, but was recovered from infected stem plates before decay and from the bases of bulb scales and leaf sheaths at the onset of bulb rot. There was no pectic enzyme activity in uninvaded onion tissue. Spread of the fungus and pectic enzyme production in two Caledon Globe genotypes susceptible or tolerant to F. oxysporum f. sp. cepae were similar, but the onset of bulb rot in tolerant genotypes was considerably delayed.     

Changes of 5＇ Terminal Nucleotides of PCR Primers Causing Variable T-A Cloning Efficiency

T-A cloning takes advantage of the unpaired adenosyl residue added to the 3＇ terminus of amplified DNAs by Taq and other thermostable DNA polymerase and uses a Ilnearlzed plasmld vector with a protruding 3＇ thymldylate residue at each of Its 3＇ termini to clone polymerase chain reaction （PCR）-derived DNA fragments. It Is a simple, reliable, and efficient Ilgatlon-dependent cloning method for PCR products, but the drawback of variable cloning efficiency occurs during application. In the present work, the relationship between variable T-A cloning efficiency and the different 5＇ end nucleotlde base of primers used In PCR amplification was studied. The results showed that different cloning efficiency was obtained with different primer pairs containing A, T, C and G at the 5＇ terminus respectively. The data shows that when the 5＇ end base of primer pair was adenosyl, more white colonies could be obtained In cloning the corresponding PCR product In comparison with other bases. And the least white colonies were formed when using the primer pair with 5＇ cytldylate end. The gluanylate end primers resulted In almost the same cloning efficiency In the white colonies amount as the thymldylate end primer did, and this efficiency was much lower than that of adenosyl end primers. This presumably is a consequence of variability In 3＇dA addition to PCR products mediated by Taq polymerase. Our results offer instructions for primer design for researchers who choose T-A cloning to clone PCR products.     

用差异显示反转录PCR银染技术研究植物基因表达的差异

通过调整差异显示反转录PCR(DDRT-PCR)中总RNA、锚定引物、随机引物、cDNA和dNTP等关键试剂的用量,优化了适用于银染检测的DDRT-PCR方法.PCR扩增产物经6%变性聚丙烯酰胺凝胶垂直电泳分离后,银染能检测到多而清晰的条带.泳道中的条带数最少为40个,最多达80个,平均为60个,条带大小分布在100～900 bp范围,灵敏度为5 pg/mm² .此方法操作简便快速,灵敏度高,重复性好.采用这个改良的方法,分析了拟南芥野生型和ast突变型基因表达的差异.从16 000个cDNA扩增产物条带中筛选出28个差异条带.二次PCR扩增后,进一步筛选出13个差异条带,其中7个是野生型特异表达的,6个是突变型特异表达的,为进一步认识ast突变表型的产生机制奠定了基础.     

Aster Yellows Phytoplasma Identified in Scentless Chamomile by Microscopical Examinations and Molecular Characterization

Scentless chamomile (Matricaria perforata Mérat) plants were commonly found infected with a yellows-type disease caused by phytoplasma in several fields in Alberta, Canada. Typical phytoplasmas were detected in the phloem cells in ultrathin sections from leaf, stem, root and flower petiole tissues examined by electron microscopy. Application of 4′6-diamidino-2-phenylindole- 2HCl (DAPI) staining techniques confirmed the presence of the phytoplasma in these tissues. These observations were supported by polymerase chain reaction (PCR) assays, using two primer pairs, P1/P6 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. Aster yellows and potato witches′-broom (PWB) DNA phytoplasma samples served as positive controls and were used to study group relatedness. In a direct PCR assay, DNA amplification with universal primer pair P1/P6 gave the expected PCR products of 1.5 kb. Based on a nested-PCR assay using the latter PCR products, as templates, and a specific primer pair R16(1)F1/R1 designed on the basis of AY phytoplasma rDNA sequences, a PCR product of 1.1 kb was obtained from each phytoplasma-infected chamomile and AY samples but not from PWB phytoplasma and healthy chamomile controls. DNA amplification with specific primer pair R16(1)F1/R1 and restriction fragment length polymorphism indicated the presence of AY phytoplasma in the infected scentless chamomile sample.     

A PCR-based diagnostic tool for distinguishing grape skin color mutants

Berry skin color mutants are phenotypically different from their original cultivars, but they show identical molecular profile if analysed by using microsatellite markers. This work gives an easy, inexpensive and quick diagnostic tool to discriminate these somatic variants. We distinguished some grape (Vitis vinifera L.) skin color mutants from white to red or pink and from black to grey, pink or white and we investigated their molecular bases by single-strand conformational polymorphism (SSCP), single base primer extension and coding sequence analysis of anthocyanin biosynthetic enzyme genes and by polymerase chain reaction (PCR) analysis of VvmybA1 regulatory gene. Analyses of structural genes did not reveal polymorphisms between wild type and mutant cultivars but only among different varieties, whereas the study of VvmybA1 regulatory gene has given important outcomes for color mutants characterisation. The discrimination between white wild type and its derived colored mutant and between black wild type and white mutant has been obtained through a simple test of amplification for presence/absence. The discrimination between black wild type and less colored mutant has occurred through a quantitative result on agarose gel confirmed by real-time PCR analysis: the amount of functional allele in less colored somatic variants genome was about one-fourth of the correspondent quantity in original black cultivars genome.     

连作花魔芋软腐病株与健株根域丛枝菌根真菌群落多样性

【目的】解析不同连作年限花魔芋软腐病株、健株根域的丛枝菌根真菌(arbuscular mycorrhizal fungi,AMF)群落多样性。【方法】使用AMF 18S SSU rRNA基因特异引物AMV4.5NF/AMDGR对正茬及连作2年和3年的软腐病株、健株魔芋根系和根际土壤DNA扩增建库,通过高通量测序和生物信息学分析探究魔芋软腐病与其根域AMF群落多样性的关系。【结果】魔芋根系具有明显的AMF菌丝、泡囊和丛枝等结构。在相同连作年限条件下,健株根系AMF总侵染率、侵染强度和孢子密度均显著高于病株(P<0.05);在不同连作年限条件下,病株根系AMF总侵染率和侵染强度随连作年限延长而降低。从所有样品中共鉴定到9属53种AMF,其中有49个已知种和4个新种。球囊霉属(Glomus)和类球囊霉属(Claroideoglomus)是AMF群落的优势属,其AMF种分别占总AMF种数的41.5%和26.4%;丰度最高的Paraglomus sp.VTX00308是所有样品的共有种。连作、软腐病及二者的交互作用显著影响根系AMF群落的Shannon指数和Simpson指数及根际土壤AMF的Chao1指数(P<0.05)。通过丰度差异分析发现6个在连作软腐病发生后丰度差异显著的AMF种(P<0.05);NMDS分析表明,不同连作年限的魔芋软腐病株与健株之间的根域AMF菌种组成、相对丰度和群落结构存在差异。相关性分析表明,软腐病发病率和病情指数与魔芋根系和根际土壤AMF的Shannon指数、根系AMF的Chao1和Simpson指数以及AMF总侵染率、侵染强度和孢子密度极显著负相关(P<0.01)。【结论】比对健株,连作魔芋软腐病株根际土壤AMF孢子密度以及根系AMF侵染率、种数和多样性均降低,其群落结构显著改变。     

Use of RAPD markers to screen somatic hybrids between Solanum tuberosum and S. brevidens

Summary The identification of somatic hybrids between Solanum tuberosum and S. brevidens can be carried out using polymerase chain reaction (PCR) and arbitrary 10-mer primers to generate random amplified polymorphic DNA (RAPD) markers. Five commercial primers have been tested. Each primer directed the amplification of a genome-specific fingerprint for the fusion parents and S. brevidens. The size of the amplified DNA fragments ranged from 100 to 1800 base pairs. The somatic hybrids showed a combination of the parental banding profiles with four of the five primers surveyed, whereas regenerants from one of the parents had the same or a similar banding pattern to that of the parent. Thus RAPD markers provide a quick, simple and preliminary screening method for putative somatic hybrids.Abbreviations EDTA ethylenediaminetetraacetic acid, - PCR polymerase chain reaction - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphisms - TBE Tris-borate-EDTA buffer - Tris trizma base     

An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules

Toward the expansion of the genetic alphabet, we present an unnatural base pair system for efficient PCR amplification, enabling the site-specific incorporation of extra functional components into DNA. This system can be applied to conventional PCR protocols employing DNA templates containing unnatural bases, natural and unnatural base triphosphates, and a 3′→5′ exonuclease-proficient DNA polymerase. For highly faithful and efficient PCR amplification involving the unnatural base pairing, we identified the natural-base sequences surrounding the unnatural bases in DNA templates by an in vitro selection technique, using a DNA library containing the unnatural base. The system facilitates the site-specific incorporation of a variety of modified unnatural bases, linked with functional groups of interest, into amplified DNA. DNA fragments (0.15 amol) containing the unnatural base pair can be amplified 10⁷-fold by 30 cycles of PCR, with <1% total mutation rate of the unnatural base pair site. Using the system, we demonstrated efficient PCR amplification and functionalization of DNA fragments for the extremely sensitive detection of zeptomol-scale target DNA molecules from mixtures with excess amounts (pmol scale) of foreign DNA species. This unnatural base pair system will be applicable to a wide range of DNA/RNA-based technologies.     

Investigations on the causes of upper stem rot (USR) on standing mature oil palms

Three different trials to examine the cause of upper stem rot (USR) infection in oil palm failed to achieve any infection. In the first experiment, inoculum was applied as colonised rubber wood blocks or as spore suspensions. In the second experiment, particular attention was given to ensure that the Ganoderma spores were freshly collected to maintain viability but no infection was observed around the inoculation sites of any of the different oil palm tissues treated. Lastly in the third experiment, both monokaryotic and dikaryotic mycelial cultures were applied directly to cut fronds, which were protected with a moist covering, but no infection was detected after more than two years. Failure to achieve infection by direct inoculation would indicate that USR does not arise from direct infection of living tissues by Ganoderma spores or mycelium, this is probably because of insufficient inoculum potential to cause infection. It is suggested that USR infection is achieved only when a sufficiently large source of inoculum has built up in dead material, probably in frond axils, and this allows invasion of the living tissues.     

PCR-based identification of microcystin-producing genotypes of different cyanobacterial genera

Microcystins are harmful hepatotoxins produced by many, but not all strains of the cyanobacterial genera Anabaena, Microcystis, Anabaena, Planktothrix, and Nostoc. Waterbodies have to be monitored for the mass development of toxic cyanobacteria; however, because of the close genetic relationship of microcystin-producing and non-producing strains within a genus, identification of microcystin-producers by morphological criteria is not possible. The genomes of microcystin-producing cells contain mcy genes coding for the microcystin synthetase complex. Based on the sequence information of mcy genes from Microcystis and Planktothrix, a primer pair for PCR amplification of a mcyA gene fragment was designed. PCR with this primer pair is a powerful means to identify microcystin-producing strains of the genera Anabaena, Microcystis, and Planktothrix. Moreover, subsequent RFLP analysis of the PCR products generated genus-specific fragments and allowed the genus of the toxin producer to be identified. The assay can be used with DNA from field samples.Abbreviations RFLP Restriction fragment length polymorphism - MALDI-TOF Matrix-assisted laser desorption/ionization-time of flight spectrometry - HPLC High performance liquid chromatography     

Identification of sex in Simmondsia chinensis (Jojoba) using RAPD markers

Simmondsia chinensis (Link) Schneider, a multipurpose dioecious shrub of arid zones, has emerged as a cash crop. It is being cultivated for its seeds which store liquid wax whose properties are similar to spermaceti (Sperm whale oil), a substitute for petro products and precious high-priced lubricants. Jojoba is a slow-growing desert shrub having a male biased (5:1; male:female ratio) population. Since there is no method available to determine the sex at the seedling stage, current investigations have been carried out to generate a sex-specific random amplified polymorphic DNA (RAPD) marker in jojoba which is based on the PCR amplification of random locations in the genome of plant. Of the 72 primers tested, only one random decamer primer, OPG-5, produced a unique ∼1,400 base pairs fragment in male DNA. To validate this observation, this primer was re-tested with the individuals of male and female samples of four cultivars. The unique ∼1,400 bp fragment was present in male individuals of all the four cultivars and completely absent in respective female individuals tested. To the best of our knowledge, this is the first report to ascertain the sex of jojoba plants at an early stage of development of the taxon.     

Possible sources of genetic resistance in oil palm (<Emphasis Type="Italic">Elaeis guineensis</Emphasis> Jacq.) to basal stem rot caused by <Emphasis Type="Italic">Ganoderma boninense</Emphasis> – prospects for future breeding

Oil palm estates in southeast Asia suffer from substantial losses due to basal stem rot caused by Ganoderma boninense. Field observations have been carried out in North Sumatra, Indonesia, on a series of planting materials of known origin. Differences in susceptibility to the disease have been detected within the two Elaeis species, guineensis and oleifera. Within Elaeis guineensis, material of Deli origin is highly susceptible compared to material of African origin. It is also possible to detect differences in reaction between parents and between crosses within a given origin. The variability of resistance to basal stem rot within the same cross is also illustrated by the diverse responses of clones derived from palms of the same origin. The prospects opened up by these results are discussed, and the importance of performing an early selection test is highlighted.     

Polymerase chain reaction analysis of transgenic plants contaminated byAgrobacterium

Agrobacterium-mediated genetic transformation is a method of choice for the development of transgenic plants. The presence of latentAgrobacterium that multiplies in the plant tissue in spite of antibiotic application confounds the results obtained by polymerase chain reaction (PCR) analysis of putative transgenic plants. The presence ofAgrobacterium can be confirmed by amplification of eitherAgrobacterium chromosomal genes or genes present out of transfer DNA (T-DNA) in the binary vector. However, the transgenic nature ofAgrobacterium-contaminated transgenic plants cannot be confirmed by PCR. Here we report a simple protocol for PCR analysis ofAgrobacterium-contaminated transgenic plants. This protocol is based on denaturation and renaturation of DNA. The contaminating plasmid vector becomes double-stranded after renaturation and is cut by a restriction enzyme having site(s) within the PCR amplicon. As a result, amplification by PCR is not possible. The genomic DNA with a few copies of the transgene remains single-stranded and unaffected by the restriction enzyme, leading to amplification by PCR. This protocol has been successfully tested with 4 different binary vectors and 3Agrobacterium tumefaciens strains: EHA105, LBA4404, and GV3101.