Dissertations / Theses on the topic 'Seed-borne plant diseases'

Copies of author's previously published articles inserted. Bibliography: leaves 128-143. This thesis describes the development of serological and nucleic acid based diagnostic methods for pea-seed borne mosaic virus (PSbMV), the isolation of specific effects on infected pea plants, the collection and biological comparison of new PSbMV isolates from Pakistan, the cloning and sequencing of specific parts of the genome of selected isolates, nucleotide and amino acid sequence comparisons between selected isolates, and the development of a ribonuclease protection assay (RPA) for identifying genomic differences among the PSbMV isolates. It is the first comparison of a range of geographically different isolates of PSbMV on the basis of both biological and molecular properties.

Corrigendum inserted at the back. Includes bibliographical references (leaves 133-158). Ch. 1. General introduction -- ch. 2. General materials and methods -- ch. 3. Biological characterisation of Australian PSbMV isolates -- ch. 4. Developing nucleic acid based diagnostics for PSbMV -- ch. 5. Detection of PSbMV isolates by RT-PCR and RFLP analysis -- ch. 6. Developing an internal control for PSbMV RT-PCR -- ch. 7. Molecular analysis of the PSbMV VPG -- ch. 8. PSbMV sequence and phylogenetic analysis -- ch. 9. General discussion Sixteen pea seed borne mosaic virus (PSbMV) isolates were collected between 1995 and 1998. These isolates were biologically distinct yet serologically indistinguishable. The conclusion is that PSbMV is widespread and occurs at a low incidence in Australia. Reports sequence information on new isolates of PSbMV which has allowed genomic regions to be identified which distinguish PSbMV pathotypes and isolates; and, to the development of PSbMV nucleic acid hybridisation and RT-PCR assays.

Resumo: Dos vírus que infectam o maracujazeiro no Brasil, atualmente o Cowpea aphid borne mosaic virus (CABMV), é considerado fator limitante à cultura. Dependendo da velocidade de disseminação e idade com que as plantas são infectadas no campo, a cultura torna-se comercialmente improdutiva. O presente estudo teve como objetivo, avaliar a diversidade e a dinâmica populacional dos afídeos na região da Alta Paulista, SP e a possibilidade de transmissão do vírus pela semente. Assim, quatro locais (Leste e Oeste da cidade de Marília e Municípios de Ocauçú e Guaimbê) foram monitorados durante 24 meses com armadilhas amarelas de água do tipo Moericke. Constatou-se nas quatro regiões a predominância do gênero Aphis. Outras espécies coletadas foram Myzus persicae, Geopenphigus flocculosus, Brevicoryne brassicae, Rhopalosiphum spp, Dysaphis spp e Lipaphis erysimi. A flutuação populacional de formas aladas do gênero Aphis, caracterizou-se por apresentar maiores revoadas em maio, junho, agosto e setembro. As espécies de Aphis (A. fabae, A. gossypii, A. spiraecola) devem ser os principais vetores do CABMV na região. Plantios novos, ao lado de plantações infectadas, tornam-se infectadas em três meses. Nos testes de transmissão através de sementes, do total de 13056 semeadas oriundas de plantas doentes, germinaram 10592, e em avaliações visuais dois meses após a germinação, não foram observadas plantas sintomáticas, indicando a não transmissão pela semente.
Abstract: From the viruses were described infecting passionfruit plants in Brasil, and the Cowpea aphid borne mosaic virus (CABMV), is considered the most hazardous. Depending on the spread velocity of aphids and the age that the plants are infected, the crops doesn’t produce commercial fruits. The present study was designed to evaluate the diversity and dynamic population of aphids in the Alta Paulista, SP region and aspects of seed transmission. For this, four regions (East and West of Marília city, Guaimbê and Ocauçú) were monitored for 24 months using yellow water Moerick trap. The predominance of the genus Aphis was observed in the four evaluated areas. Other species founded in the area were: Myzus persicae, Geopenphigus flocculosus, Brevicoryne brassicae, Rhopalosiphum spp, Dysaphis spp and Lipaphis erysimi. The population curve of alate Aphis spp showed the highest frequency of flights during May, June, August and September. The Aphis spp (A. fabae, A. gossypii, A. spiraecola) probably is the most important vector of the CABMV in the region. New crops near old infected plants, were infected in three months. To evaluate properties of seed transmission, from 13056 collected from infected plants, 10592 were germinated and evaluated during two months for the presence of visual symptoms. No plants with simptoms were observed indicating no seed transmission.
Orientador: Marcelo Agenor Pavan
Coorientador: Valdir Atsushi Yuki
Banca: Renate Krause Sakate
Banca: Aloisio Costa Sampaio
Banca: Alexandre Levi R. Chaves
Banca: Hugo Kuniyuki
Doutor

Thesis (PhDAgric (Plant Pathology))--Stellenbosch University, 2010.
ENGLISH ABSTRACT: In the Western Cape onion industry in South Africa, Fusarium oxysporum Schlechtend.:Fr. f.sp. cepae (H.N. Hans.) W.C. Snyder & H.N. Hans. (Focep) has been identified as the leading cause of harvest and storage losses. This pathogen is of world-wide importance and causes Fusarium basal rot of onions (Allium cepa), affecting all onion growth stages. No information is available on the evolution, genetic diversity, molecular detection and inoculum sources of the South African Focep population. Similar to what is the case for South Africa, limited information is available on Focep in other regions of the world. World-wide, four vegetative compatibility groups (VCGs) and two single-member VCGs (SMVs) have been identified among two Japanese and 19 Colorado (USA) isolates. This polyphyletic origin of Focep suggested by VCG analyses was confirmed through molecular analyses of isolates from a few countries. Only the mating type (MAT)1-1 idiomorph has been reported for Focep isolates from Welsh onion (Allium fistulosum). The development of sustainable management strategies of Focep is dependent on knowledge of (i) the genetic diversity and evolution of Focep, (ii) whether high throughput molecular methods can be developed for identifying the most virulent and widespread Focep genotypes and (iii) the role of seedlings and seeds as primary inoculum sources, and the Focep genotypes associated with these growth stages. Therefore, the three main aims of the current study were to investigate the aforementioned three aspects. In the first aim of the study, the genetic diversity and evolution of Focep was investigated using a collection of 79 F. oxysporum isolates from South Africa (27 Focep and 33 non-pathogenic isolates) and Colorado (19 Focep isolates). VCG analyses revealed the presence of six VCGs, four among the Colorado Focep isolates (VCGs 0421, 0422, 0423 and 0424) and two among the South African bulb-associated isolates (VCGs 0425 and 0426). VCG 0421 and VCG 0425 were the two main VCGs in Colorado and South Africa, respectively. Four SMVs and one heterokaryon selfincompatible (HSI) isolate were also identified. The polyphyletic nature of Focep in South Africa and Colorado was shown through a combined translation elongation factor 1α (EF-1α) and mitochondrial small-subunit (mtSSU) phylogeny. The phylogeny divided the Focep isolates into two main clades, of which one contained the two main VCGs (0421 and 0425), SMVs and non-pathogenic isolates. The second, ancestral clade contained the HSI isolate, VCGs 0422, 0423 and 0424, and non-pathogenic isolates. Unlike the clade containing the two main VCGs, which were highly virulent toward onion bulbs, the ancestral clade contained isolates that were mostly moderately virulent. The incongruence of the EF-1α and mtSSU datasets with an intergenic spacer (IGS) region data set, and the presence of both MAT idiomorphs within the same isolate for some isolates, suggested possible exchange of genetic material between isolates. The second aim of the study was to develop molecular methods for identifying the two main Focep VCGs (0425 and 0421), using DNA fingerprinting methods and sequence-characterized amplified region (SCAR) markers. These techniques were first developed using the F. oxysporum isolates from the first aim, and were then used to investigate the prevalence of VCG 0425 among 88 uncharacterized F. oxysporum isolates from onion bulbs in South Africa. Two random amplified polymorphic DNA primers provided two diagnostic amplicons for VCG 0425, but attempts to develop SCAR markers from these amplicons were unsuccessful. In contrast, an interretrotransposon amplified polymorphism (IRAP) fingerprinting method enabled the developed of a multiplex IR-SCAR polymerase chain reaction method that detected the VCG 0421, 0425 and SMV 4 isolates as a group. Fingerprinting and SCAR marker testing of the 88 uncharacterized F. oxysporum isolates from South Africa (65 Focep and 23 non-pathogenic) confirmed that VCG 0425 is the main VCG in South Africa associated with mature onion bulbs, since 63 of the Focep isolates had the molecular characteristics of VCG 0425. The third aim of the study was to determine whether seed and seedling transplants are inoculum sources of Focep, and whether the same genotype (VCG 0425) that dominated on mature bulbs could be detected from these sources. Focep isolates were obtained from seven of the 13 investigated onion seed lots, as well as from onion seedling transplants that were collected from all five onion nurseries in the Western Cape. Focep seedling infection more than doubled from the 6-week growth stage to the 14-week growth stage. Seed infections by Focep were low, but the seedborne nature of Focep was confirmed by showing that a green fluorescent protein labelled Focep transformant could be transmitted from infected soil to onion seed via the onion bulbs and seedstalks. It is thus clear that commercial seed and seedlings are inoculum sources of Focep. However, the Focep genotypes on seed and seedlings are different from those in mature bulbs and were not dominated by VCG 0425. Furthermore, most (≤ 60%) of the seed and seedling isolates were moderately virulent, as compared to the mostly highly virulent isolates from mature bulbs.
AFRIKAANSE OPSOMMING: In die Wes-Kaapse uiebedryf in Suid-Afrika is Fusarium oxysporum Schlechtend.:Fr. f.sp. cepae (H.N. Hans.) W.C. Snyder & H.N. Hans. (Focep) geïdentifiseer as die vernaamste oorsaak van oes- en opbergingsverliese. Hierdie patogeen is van wêreldwye belang; dit veroorsaak Fusarium-bolvrot van uie (Allium cepa) en affekteer alle plantgroeistadia. In Suid-Afrika is daar geen inligting beskikbaar oor die evolusie, genetiese diversiteit, molekulêre opsporing en inokulumbronne van die Focep-populasie nie. Soortgelyk aan wat die geval in Suid-Afrika is, is daar beperkte inligting beskikbaar oor Focep in ander wêrelddele. Wêreldwyd is daar vier vegetatiewe versoenbaarheidsgroepe (VVGe) en twee enkellid VVGe (ELVe) geïdentifiseer onder twee Japannese en 19 Colorado (VSA) isolate. Hierdie veelvuldige oorsprong van Focep wat deur VVG-analise voorgestel was, is deur die molekulêre analises van isolate uit ’n paar ander lande bevestig. Slegs die paringstipe (PT)1-1 idiomorf is vir Focep-isolate uit Walliese-tipe uie (ook bekend as ‘lenteuie’ in Suid Africa) (Allium fistulosum) berig. Die ontwikkeling van volhoubare bestuurstrategieë vir Focep steun op kennis van (i) die genetiese diversiteit en evolusie van Focep, (ii) of hoë-deurset molekulêre metodes ontwikkel kan word vir die identifisering van die mees virulente en wydverspreide Focep-genotipes en (iii) die rol van saailinge en saad as primêre inokulumbronne, en die Focep-genotipes wat met hierdie groeistadia geassosieer word. Daarom was die hoof doelstellings van hierdie studie om die bogenoemde drie aspekte te bestudeer. Om die eerste doel van die studie te bereik is die genetiese diversiteit en evolusie van Focep bestudeer deur gebruik te maak van ‘n versameling van 79 F. oxysporum-isolate uit Suid-Afrika (27 Focep en 33 nie-patogeniese isolate) en uit Colorado (19 Focep-isolate). VVG-analises het die teenwoordigheid van ses VVGe aangetoon – vier onder die Colorado Focep-isolate (VVGe 0421, 0422, 0423 en 0424) en twee onder die Suid-Afrikaanse bol-geassosieerde isolate (VVGe 0425 en 0426). VVG 0421 en VVG 0425 was die twee hoof VVGe in onderskeidelik Colorado en Suid-Afrika. Vier ELVe en een meerkernige self-onversoenbare (MSO) isolaat is ook geïdentifiseer. Die veelvuldige oorsprong van Focep in Suid-Afrika en Colorado is ook aangetoon deur ‘n gekombineerde translasie verlengings faktor 1α (VF-1α) en mitokondriale klein-subeenheid (mtKSE) filogenie. Dié filogenie het die Focepisolate in twee groepe verdeel, waarvan die een groep die twee hoof VVGe (0421 en 0425), ELVe en nie-patogeniese isolate bevat het. Die tweede, basal groepering het die MSO-isolaat, VVGe 0422, 0423 en 0424, en nie-patogeniese isolate bevat. In teenstelling met die eersgenoemde groepering wat hoogs virulente isolate van uiebolle bevat het, het die basale groepering isolate bevat wat meestal matig virulent was. Die inkongruensie van die VF-1α en mtKSE-datastelle met ‘n intergeen-gespasieerde (IGS) area datastel – asook die teenwoordigheid van beide PT-idiomorwe binne dieselfde isolaat by sommige isolate – het op ’n moontlike uitruiling van genetiese materiaal tussen isolate gedui. Die tweede doel van die studie was om molekulêre metodes te ontwikkel vir die identifisering van die twee hoof Focep VVGe (0425 en 0421) deur gebruik te maak van DNA-vingerafdrukke en nukleotied-gekarakteriseerde geamplifiseerde area (NKAA) merkers. Hierdie tegnieke is ontwikkel deur van die F. oxysporum-isolate van die eerste doelstelling gebruik te maak en is daarna gebruik om die frekwensie van VVG 0425 onder 88 ongekarakteriseerde F. oxysporum-isolate van uiebolle in Suid-Afrika te ondersoek. Twee gerandomiseerde geamplifiseerde polimorfiese DNS (RAPD) merkers het twee diagnostiese nukleotiedbasis-areas vir VVG 0425 gelewer, maar pogings om NKAA-merkers uit hierdie geamplifiseerde nukleotiedbasis-areas te onwikkel was onsuksesvol. In teenstelling hiermee het ‘n inter-retrotransposon geamplifiseerde polimorfisme (IRAP) vingerafdrukmetode die ontwikkeling van ‘n multipleks IR-NKAA polimerase kettingreaksiemetode moontlik gemaak wat die VVG 0421-, VVG 0425- en ELV 4-isolate as ’n groep aangedui het. Vingerafdruktoetsing en NKAA-merkertoetsing van die 88 ongekaraktariseerde F. oxysporum isolate van Suid-Afrika (65 Focep en 23 nie-patogenies) het bevestig dat VVG 0425 die hoof VVG in Suid-Afrika is wat met volwasse bolle geassosieer word, aangesien 63 van die Focep-isolate die molekulêre eienskappe van VVG 0425 gehad het. Die derde doel van die studie was om vas te stel of saad en saailinge inokulumbronne van Focep is, en of dieselfde genotipe (VVG 0425) wat op volwasse bolle dominant is, waargeneem kon word op hierdie bronne. Focep-isolate is verkry van sewe van die 13 uiesaadlotte asook van uiesaailinge wat in al vyf uiesaailingkwekerye in die Wes-Kaap versamel is. Focep-saailinginfeksie was meer as dubbel in die 14-week groeistadium as wat dit in die 6-week stadium was. Saadinfeksies deur Focep was laag, maar die saadgedraagde aard van Focep is bevestig deur aan te toon dat ’n Focep-transformant wat met ‘n groen fluoreserende proteïen geëtiketeer is, van geïnfekteerde grond na uiesaad oorgedra kon word via die uiebolle en -saadstele. Dit is dus duidelik dat kommersiële saad en saailinge as inokulumbronne van Focep dien. Die Focep-genotipes op saad en saailinge verskil egter van dié in volwasse bolle en is nie deur VVG 0425 gedomineer nie. Verder was die meeste (≤ 60%) saad- en saailingisolate matig virulent, in teenstelling met die meestal hoogs virulente isolate uit volwasse bolle.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Dos vírus que infectam o maracujazeiro no Brasil, atualmente o Cowpea aphid borne mosaic virus (CABMV), é considerado fator limitante à cultura. Dependendo da velocidade de disseminação e idade com que as plantas são infectadas no campo, a cultura torna-se comercialmente improdutiva. O presente estudo teve como objetivo, avaliar a diversidade e a dinâmica populacional dos afídeos na região da Alta Paulista, SP e a possibilidade de transmissão do vírus pela semente. Assim, quatro locais (Leste e Oeste da cidade de Marília e Municípios de Ocauçú e Guaimbê) foram monitorados durante 24 meses com armadilhas amarelas de água do tipo Moericke. Constatou-se nas quatro regiões a predominância do gênero Aphis. Outras espécies coletadas foram Myzus persicae, Geopenphigus flocculosus, Brevicoryne brassicae, Rhopalosiphum spp, Dysaphis spp e Lipaphis erysimi. A flutuação populacional de formas aladas do gênero Aphis, caracterizou-se por apresentar maiores revoadas em maio, junho, agosto e setembro. As espécies de Aphis (A. fabae, A. gossypii, A. spiraecola) devem ser os principais vetores do CABMV na região. Plantios novos, ao lado de plantações infectadas, tornam-se infectadas em três meses. Nos testes de transmissão através de sementes, do total de 13056 semeadas oriundas de plantas doentes, germinaram 10592, e em avaliações visuais dois meses após a germinação, não foram observadas plantas sintomáticas, indicando a não transmissão pela semente.
From the viruses were described infecting passionfruit plants in Brasil, and the Cowpea aphid borne mosaic virus (CABMV), is considered the most hazardous. Depending on the spread velocity of aphids and the age that the plants are infected, the crops doesn t produce commercial fruits. The present study was designed to evaluate the diversity and dynamic population of aphids in the Alta Paulista, SP region and aspects of seed transmission. For this, four regions (East and West of Marília city, Guaimbê and Ocauçú) were monitored for 24 months using yellow water Moerick trap. The predominance of the genus Aphis was observed in the four evaluated areas. Other species founded in the area were: Myzus persicae, Geopenphigus flocculosus, Brevicoryne brassicae, Rhopalosiphum spp, Dysaphis spp and Lipaphis erysimi. The population curve of alate Aphis spp showed the highest frequency of flights during May, June, August and September. The Aphis spp (A. fabae, A. gossypii, A. spiraecola) probably is the most important vector of the CABMV in the region. New crops near old infected plants, were infected in three months. To evaluate properties of seed transmission, from 13056 collected from infected plants, 10592 were germinated and evaluated during two months for the presence of visual symptoms. No plants with simptoms were observed indicating no seed transmission.

The limited specificity, sensitivity and multiplex capacity of detection techniques currently available for important seed-borne pathogens of tomato is a significant risk for the global tomato trade and production industry. These pathogens can be associated with seed at low concentrations but, due to their highly virulent nature, these low levels can be sufficient to infect germinating seedlings and spread to neighbouring plants and fields, potentially causing epidemics and economic losses. In this study, detection techniques currently available for phytodiagnostics were evaluated for the capacity to accurately detect and identify five agronomically important seed-borne pathogens of tomato: Pepino mosaic virus (PepMV), Tomato mosaic virus (ToMV), Clavibacter michiganensis subsp. michiganensis (Cmm), Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato. A prototype diagnostic microarray was also designed in an attempt to develop a tool that could simultaneously detect these five seed-borne pathogens from a single sample. Viral detection based on serological techniques was rapid, accurate and reliable but only detected a single pathogen per assay and required supplementary bioassays to indicate the viability of detected viral pathogens. Selective media plating for bacterial detection demonstrated unreliable recovery of targeted bacteria from infected seed and leaf samples and required supplementary tests to validate the identity of presumptive positives. Assays were lengthy, laborious and sometimes too ambiguous for accurate diagnosis of bacterial pathogens. Nucleic acid-based technologies demonstrated improved sensitivity and specificity for detection of targets from pure culture, leaf and seed extracts, compared to conventional and serological methods, yet also required supplementary bioassays or media assays to validate the viability of detected pathogens. Amplification efficiency however, was affected by the presence of PCR inhibitors and despite positive detection, variable banding intensity in electrophoretic analysis of amplified products necessitated the use of reference cultures to validate diagnosis. The developed microarray incorporated 152 pathogen-specific and control probes to facilitate diagnosis and taxonomic classification of detected pathogens. The array was challenged with pure culture extracts of the five target pathogens, selected related and non-target, unrelated pathogens of tomato. Positive detection of each of the pathogens was demonstrated but the production of hybridisation signals was highly variable and extremely sensitive to minor technical differences. Each of the five pathogens were successfully detected in combination proving that different classes of seed-borne pathogens could be detected from a single sample using the developed microarray. This prototype microarray has good potential for phytodiagnostic screening of the five targeted pathogens, and further validation, optimisation and extension for testing tomato seed samples may facilitate incorporation of this array into standard diagnostic protocols.

Seed piece to plant transmission of the potato late blight pathogen, Phytophthora infestans, occurred with isolates of the clonal lineages US-8 in Oregon and US-11 in Washington in field trials. Average transmission rate across potato cultivars was 0.5 and 2.4% with US-8, and 0.8 and 1.0% with US-11 in 1999 and 2000, respectively. Transmission rate with US-8 was 2.3% for Russet Burbank (RB) in 1999 and 1.7, 0.7, 4.3, 7.6 and 0.5% for Bannock, Bzura, Ranger, Russet Norkotah (RN), and Umatilla, respectively, in 2000. Transmission rate with US-11 in 1999 was 0.5, 4.9 and 1.4% for RB, RN, and Shepody, respectively, and 1.7% for RB in 2000. Seedborne inoculum of both clonal lineages significantly affected stand establishment and plant vigor. With US-8, final emergence, emergence rate, and aerial biomass of cvs Kennebec, RB, RN, and Shepody were significantly lower than Bzura in 1999, whereas in 2000, these same responses in Chieftain, Bannock, Ranger, and Shepody were significantly lower than Bzura, Umatilla and RN. With US-11, these same response variables were significantly lower in Kennebec, RN and Shepody compared to Bzura and RB in 1999, and were significantly lower in Bannock, Chieftain, Ranger and Shepody compared to RB and Umatilla in 2000. Plant growth responses of cvs RB and RN grown from seed pieces infected with US-8 or US-11 were evaluated in greenhouse trials. RN was equally susceptible to both clonal lineages whereas RB was more resistant than RN to seedborne inoculum of US-11. Compared to RN its final emergence was higher, emergence rate was faster, aerial biomass was greater, and seed piece decay was lower. US-8 was more aggressive than US-11 on RB. US-8 caused a greater reduction in final emergence, emergence rate, and aerial biomass, and a greater increase in seed piece decay. The two clonal lineages were similar in their aggressiveness on RN. This is the first report of cultivar*clonal lineage*inoculum density interactions for plant growth responses of potato grown from seed pieces infected with P. infestans.
Graduation date: 2002

Copies of author's previously published articles inserted.
Bibliography: leaves 128-143.
xi, 143, [44] leaves, [36] leaves of plates : ill. (some col.) ; 30 cm.
This thesis describes the development of serological and nucleic acid based diagnostic methods for pea-seed borne mosaic virus (PSbMV), the isolation of specific effects on infected pea plants, the collection and biological comparison of new PSbMV isolates from Pakistan, the cloning and sequencing of specific parts of the genome of selected isolates, nucleotide and amino acid sequence comparisons between selected isolates, and the development of a ribonuclease protection assay (RPA) for identifying genomic differences among the PSbMV isolates. It is the first comparison of a range of geographically different isolates of PSbMV on the basis of both biological and molecular properties.
Thesis (Ph.D.)--University of Adelaide, Dept. of Crop Protection, 1999

Corrigendum inserted at the back.
Includes bibliographical references (leaves 133-158).
xvi, 158 leaves : ill., col. map ; 30 cm.
Sixteen pea seed borne mosaic virus (PSbMV) isolates were collected between 1995 and 1998. These isolates were biologically distinct yet serologically indistinguishable. The conclusion is that PSbMV is widespread and occurs at a low incidence in Australia. Reports sequence information on new isolates of PSbMV which has allowed genomic regions to be identified which distinguish PSbMV pathotypes and isolates; and, to the development of PSbMV nucleic acid hybridisation and RT-PCR assays.
Thesis (Ph.D.)--University of Adelaide, Dept. of Applied and Molecular Ecology, 2001