Identification and Genetic Division of Fusarium graminearum and Fusarium asiaticum by Species‐Specific SCAR Markers

Fusarium head blight (FHB), also called scab, is a devastating and insidious disease of cereals including wheat (Triticum spp.) and barley (Hordeum vulgare L.) worldwide. Apart from direct yield losses, the most serious concern about FHB is the contamination of the crop with mycotoxins, which pose a health risk to human and livestock. Recent research reported that phylogenetic species F. asiaticum (Fa) and F. graminearum (Fg) were the major causal agents of FHB from infected wheat heads in China. To investigate the population structure of Fusarium species in China by species‐specific as well as the chemotype‐specific markers, sequence‐related amplified polymorphism (SRAP) markers were screened on representative isolates of F. asiaticum‐NIV, F. asiaticum‐ 3ADON and F. graminearum‐15ADON to find amplification products characteristic of either species or chemotypes. Selected amplified fragments were cloned and sequenced so that sequence‐characterized amplified region (SCAR) primer pairs could be developed which permit specific detection of Fusarium species using conventional PCR. Primer pairs SCAR‐Fa1 and SCAR‐Fg1 were confirmed to be able to amplify specific products only in F. asiaticum and F. graminearum isolates, respectively. These species‐specific primers were applied to determine genetic division of F. asiaticum and F. graminearum isolates collected in Yangtze–Huaihe valley. The results indicated that F. asiaticum was the predominant species causing FHB in this wheat production area. It is the first report that SRAP markers were adapted for species characterization in Fusarium isolates.     

Expanded Strain‐Specific RT‐PCR Assay for Differential Detection of Currently Known Citrus Tristeza Virus Strains: a Useful Screening Tool

Genotypic characterization of Citrus tristeza virus (CTV) strains has progressed significantly, but their phenotypic expression is poorly established as CTV naturally occurs as mixed‐strain populations. A screening system for the analysis of mixed‐strain populations is required for population studies and the correlation with symptom expression. In this study, a published CTV strain‐specific detection assay was expanded and improved to facilitate detection of currently known CTV strains. Supplementary RT‐PCR assays were developed for two variant groups of the RB strain and the HA16‐5 strain, and assays for the T36 strain and generic CTV detection were improved. The value of the strain‐specific assays was shown by the ability to identify the strain components of two CTV cross‐protecting sources, GFMS35 and LMS6, used in the South African budwood certification scheme and to demonstrate the segregation of strains in budwood source trees.     

Development and validation of microsatellite markers for Karnal bunt (Tilletia indica) and loose smut (Ustilago segetum tritici) of wheat from related fungal species

Simple sequence repeats (SSRs) are preferred molecular markers because of their abundance, robustness, high reproducibility, high efficiency in detecting variation and suitability for high‐throughput analysis. In this study, an attempt was made to mine and analyse the SSRs from the genomes of two seed‐borne fungal pathogens, viz Ustilago maydis, which causes common smut of maize, and Tilletia horrida, the cause of rice kernel smut. After elimination of redundant sequences, 2,703 SSR loci of U. maydis were identified. Of the remaining SSRS, 44.5% accounted for di‐nucleotide repeats followed by 29.8% and 2.7% tri‐ and tetranucleotide repeats, respectively. Similarly, 2,638 SSR loci were identified in T. horrida, of which 20.2% were di‐nucleotide, 50.4% tri‐ and 20.5% tetra‐nucleotide repeats. A set of 65 SSRs designed from each fungus were validated, which yielded 23 polymorphic SSRs from Ustilago and 21 from Tilletia. These polymorphic SSR loci were also successfully cross‐amplified with the Ustilago segetum tritici and Tilletia indica. Principal coordinate analysis of SSR data clustered isolates according to their respective species. These newly developed and validated microsatellite markers may have immediate applications for detection of genetic variability and in population studies of bunt and smut of wheat and other related host plants. Moreover, this is first comprehensive report on molecular markers suitable for variability studies in wheat seed‐borne pathogens.     

Detection and Identification of Aster Yellows Phytoplasma Associated with Lipstick Yellow Frond Disease in Malaysia

Yellowing symptoms similar to coconut yellow decline phytoplasma disease were observed on lipstick palms (Cyrtostachys renda) in Selangor state, Malaysia. Typical symptoms were yellowing, light green fronds, gradual collapse of older fronds and decline in growth. Polymerase chain reaction assay was employed to detect phytoplasma in symptomatic lipstick palms. Extracted DNA was amplified from symptomatic lipstick palms by PCR using phytoplasma‐universal primer pair P1/P7 followed by R16F2n/R16R2. Phytoplasma presence was confirmed, and the 1250 bp products were cloned and sequenced. Sequence analysis indicated that the phytoplasmas associated with lipstick yellow frond disease were isolates of ‘Candidatus Phytoplasma asteris’ belonging to the 16SrI group. Virtual RFLP analysis of the resulting profiles revealed that these palm‐infecting phytoplasmas belong to subgroup 16SrI‐B and a possibly new 16SrI‐subgroup. This is the first report of lipstick palm as a new host of aster yellows phytoplasma (16SrI) in Malaysia and worldwide.     

Barcoding the kingdom Plantae: new PCR primers for ITS regions of plants with improved universality and specificity

The internal transcribed spacer (ITS) of nuclear ribosomal DNA is one of the most commonly used DNA markers in plant phylogenetic and DNA barcoding analyses, and it has been recommended as a core plant DNA barcode. Despite this popularity, the universality and specificity of PCR primers for the ITS region are not satisfactory, resulting in amplification and sequencing difficulties. By thoroughly surveying and analysing the 18S, 5.8S and 26S sequences of Plantae and Fungi from GenBank, we designed new universal and plant‐specific PCR primers for amplifying the whole ITS region and a part of it (ITS1 or ITS2) of plants. In silico analyses of the new and the existing ITS primers based on these highly representative data sets indicated that (i) the newly designed universal primers are suitable for over 95% of plants in most groups; and (ii) the plant‐specific primers are suitable for over 85% of plants in most groups without amplification of fungi. A total of 335 samples from 219 angiosperm families, 11 gymnosperm families, 24 fern and lycophyte families, 16 moss families and 17 fungus families were used to test the performances of these primers. In vitro PCR produced similar results to those from the in silico analyses. Our new primer pairs gave PCR improvements up to 30% compared with common‐used ones. The new universal ITS primers will find wide application in both plant and fungal biology, and the new plant‐specific ITS primers will, by eliminating PCR amplification of nonplant templates, significantly improve the quality of ITS sequence information collections in plant molecular systematics and DNA barcoding.     

Cytotoxic and Melanogenesis‐Inhibitory Activities of Limonoids from the Leaves of Azadirachta indica (Neem)

Seventeen limonoids (tetranortriterpenoids), 1 – 17 , including three new compounds, i.e., 17‐defurano‐17‐(2,5‐dihydro‐2‐oxofuran‐3‐yl)‐28‐deoxonimbolide ( 14 ), 17‐defurano‐17‐(2ξ‐2,5‐dihydro‐2‐hydroxy‐5‐oxofuran‐3‐yl)‐28‐deoxonimbolide ( 15 ), and 17‐defurano‐17‐(5ξ‐2,5‐dihydro‐5‐hydroxy‐2‐oxofuran‐3‐yl)‐2′,3′‐dehydrosalannol ( 17 ), were isolated from an EtOH extract of the leaf of neem (Azadirachta indica). The structures of the new compounds were elucidated on the basis of extensive spectroscopic analyses and comparison with literature. Upon evaluation of the cytotoxic activities of these compounds against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK‐BR‐3) cancer cell lines, seven compounds, i.e., 1 – 3, 12, 13, 15 , and 16 , exhibited potent cytotoxicities with IC₅₀ values in the range of 0.1–9.9 μM against one or more cell lines. Among these compounds, cytotoxicity of nimonol ( 1 ; IC₅₀ 2.8 μM ) against HL60 cells was demonstrated to be mainly due to the induction of apoptosis by flow cytometry. Western blot analysis suggested that compound 1 induced apoptosis via both the mitochondrial and death receptor‐mediated pathways in HL60 cells. In addition, when compounds 1 – 17 were evaluated for their inhibitory activities against melanogenesis in B16 melanoma cells, induced with α‐melanocyte‐stimulating hormone (α‐MSH), seven compounds, 1, 2, 4 – 6, 15 , and 16 , exhibited inhibitory activities with 31–94% reduction of melanin content at 10 μM concentration with no or low toxicity to the cells (82–112% of cell viability at 10 μM ). All 17 compounds were further evaluated for their inhibitory effects against the Epstein? Barr virus early antigen (EBV‐EA) activation induced by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in Raji cells.     

Identification and Molecular Characterizations of Neoscytalidium dimidiatum Causing Stem Canker of Red‐fleshed Dragon Fruit (Hylocereus polyrhizus) in Malaysia

During the year 2008 to 2009, a new disease of stem canker was noticed in most red‐fleshed dragon fruit (Hylocereus polyrhizus) plantations in Malaysia. The symptoms observed were small circular sunken orange spot, black pycnidia and rotted stem. This study was conducted to determine the occurrence of the stem canker on H. polyrhizus in Malaysia, subsequently to isolate, identify and characterize the fungal pathogen based on morphology and molecular characteristics and pathogenicity test. From the surveyed 20 plantations in Malaysia, stem canker was detected in all the plantations. A total of 40 isolates of Scytalidium‐like fungus were isolated and identified as Neoscytalidium dimidiatum based on morphological characteristics and ITS region sequences, which showed 99% similarity to N. dimidiatum (FJ648577). From the phylogenetic analysis using maximum‐likelihood tree, isolates of N. dimidiatum from stem canker of H. polyrhizus were grouped together and did not show any sequence variation. From pathogenicity test, all 40 isolates of N. dimidiatum were pathogenic causing stem canker on H. polyrhizus. To our knowledge, this is the first report of stem canker of H. polyrhizus caused by N. dimidiatum in Malaysia.     

A multiple PCR‐LDR assay for identification of two invasive cryptic species of whiteflies Bemisia tabaci

The MEAM1 and MED species of the cryptic species complex Bemisia tabaci are important invasive pests that cause tremendous crop losses worldwide. A rapid and highly reliable molecular technique is necessary to identify these species because they are morphologically indistinguishable. Therefore, a multiple polymerase chain reaction coupled with a ligase detection reaction (PCR‐LDR) that was based on polymorphisms in the mitochondrial cytochrome oxidase I (mtCOI) gene of B. tabaci was developed to distinguish the two cryptic species. An assessment of the method indicated that PCR‐LDR provided high specificity and sensitivity in discriminating MEAM1 (SHB) and MED (SHQ) whiteflies. In field tests, PCR‐LDR genotyping was performed in one 96‐well plate to identify 93 individuals collected from 8 districts in the suburbs of Shanghai. Complete concordance was observed between PCR‐LDR and sequencing methods. The method was used to confirm that MEAM1 and MED were found in two districts, but only the MED was found in the other six districts. PCR‐LDR, which is a transplantable platform, provides an alternative method for species identification of B. tabaci at low cost.     

Rapid detection of viable Legionella pneumophila in tap water by a qPCR and RT‐PCR‐based method

Aims

A molecular method for a rapid detection of viable Legionella pneumophila of all serogroups in tap water samples was developed as an alternative to the reference method (ISO). Legionellae are responsible for Legionnaires’ disease, a severe pneumonia in humans with high lethality.

Methods and Results

The developed method is based on a nutritional stimulation and detection of an increase in precursor 16S rRNA as an indicator for viability. For quantification, DNA was detected by qPCR. This method was compared to the ISO method using water samples obtained from public sports facilities in Switzerland. The sensitivity and specificity were 91 and 97%, respectively, when testing samples for compliance with a microbiological criterion of 1000 cell equivalents per l.

Conclusion

The new method is sensitive and specific for Leg. pneumophila and allows results to be obtained within 8 h upon arrival, compared to one week or more by the ISO method.

Significance and Impact of the Study

The method represents a useful tool for a rapid detection of viable Leg. pneumophila of all serogroups in water by molecular biology. It can be used as an alternative to the ISO method for official water analysis for legionellae and particularly when a short test time is required.     

Detection and Identification of Two Phytoplasmas (16SrIII‐B and 16SrXII‐A) From Alfalfa (Medicago sativa) in Serbia

Plants of alfalfa (Medicago sativa) exhibiting general stunting, proliferation and phyllody associated with leaf yellowing and reddening were observed in three localities of Central Serbia. Phytoplasma strains belonging to 16SrIII‐B and 16SrXII‐A groups were detected and identified by RFLP and sequence analysis of 16S rDNA. Stolbur phytoplasma tuf gene RFLP analysis showed the presence of the TufAY‐b‐type phytoplasma subgroup in 80% of symptomatic samples. This is the first report of 16SrIII‐B and 16SrXII‐A phytoplasma groups affecting alfalfa in Serbia.     

Isolation and Identification of Pathogens Causing Marigold (Tagetes erecta L.) Black Spot in Beijing,China

Black spot leads to great marigold losses worldwide. The disease is characterized by black spots on leaves and stems in its early stages, and the whole plant has black rot at the advanced stage. In this report, 6 of 217 Alternaria strains isolated from lesions of marigold plants in Beijing were randomly selected. The morphological characteristics and a pathogenic tree based on two protein‐coding genes (gpd and alt a 1) indicated that Alternaria tagetica is the causal agent of marigold black spot in Beijing. All six Alternaria strains could successfully re‐infect marigold, but they could not infect carrot or zinnia by either spore spray in a greenhouse or planting experiments in the epidemic area. This is the first report of the A. tagetica pathogen being isolated from marigold in Beijing.     

Specific and Rapid Detection of Lily symptomless virus and Arabis mosaic virus in Lily by Dual IC‐RT‐PCR

Lily symptomless virus (LSV) and Arabis mosaic virus (ArMV) cause severe losses of quantity and quality of lily flower and bulb production. Specificity, sensitivity and speed of detection methods for viruses need to be improved greatly to prevent LSV and ArMV from spreading from infected lilies. A dual IC‐RT‐PCR procedure for detection was developed in which the antibodies of LSV and ArMV were mixed and the mixture used to coat the PCR tubes. The particles of the two viruses were captured by the respective antibodies. Interference by other RNA viruses in infected lily was eliminated in the RT‐PCR. Also, an RNA extraction step was omitted. The dual IC‐RT‐PCR products of LSV and ArMV were 521 bp and 691 bp, respectively. The specificity of the method was validated; only LSV and ArMV of four viruses were detected by dual IC‐RT‐PCR. The sensitivity of the detection method is 1 mg leaf tissue and higher than DAS‐ELISA due to enrichment by dual immunocapture.