For almost 40 years, Australian researchers have been part of an international group of scientists who have studied graft-transmitted disorders of the grapevine. The Australian wine and grape industries are undergoing significant expansion as is the case in some other countries. Preventing the spread of pathogens, by producing clean propagation material, and minimising the disease load on new vines, is essential for the continuing success of the industry. This book covers the characteristics of each class of graft-transmitted pathogen, their effect on vines, how they spread and strategies for their control. Eleven of the most important diseases are illustrated and described comprehensively, including information about occurrence, symptoms, detection, transmission and effect on yield and quality. Finally there is a discussion of quarantine issues and disease management. This book will be an invaluable teaching tool and is intended for vineyard managers, grape growers, consultants, extension offers and students. While it provides a basic understanding of the nature of pathogens, it will aid in field assessment and identification of the often confusing disease symptoms.

A wide-ranging examination of plastid (pt)DNA sequence homologies within higher plant nuclear genomes (promiscuous DNA) was undertaken. Digestion with methylation-sensitive restriction enzymes and Southern analysis was used to distinguish plastid and nuclear DNA in order to assess the extent of variability of promiscuous sequences within and between plant species. Some species, such as Gossypium hirsutum (cotton), Nicotiana tabacum (tobacco), and Chenopodium quinoa showed homogenity of these sequences, while intraspecific sequence variation was observed among different cultivars of Pisum sativum (pea), Hordeum vulgare (barley), and Triticum aestivum (wheat). Hypervariability of plastid sequence homologies was identified in the nuclear genomes of Spinacea oleracea (spinach) and Beta vulgaris (beet), in which individual plants were shown to possess a unique spectrum of nuclear sequences with ptDNA homology. This hypervariability apparently extended to somatic variation in B. vulgaris. No sequences with ptDNA homology were identified by this method in the nuclear genome of Arabidopsis thaliana.

Leaves were collected from 21 different grapevine (Vitis ssp.) genotypes with varying resistance to powdery mildew disease caused by Uncinula necator. For leaves collected from field-grown vines in spring there was a correlation between resistance rating and activity of chitinase and β-1,3-glucanase. The correlation was greater with the sum of the two enzyme activities. In contrast, no correlation was obtained for leaves collected during summer. With leaves from glasshouse grown vines, wounding or infection with powdery mildew increased both chitinase and β-1,3-glucanase activity. Light microscope examination of detached leaves inoculated with U. necator conidia showed that germination appeared to occur at the same rate on leaves of a susceptible (Sultana) and a resistant (Seyval) genotype. Subsequent development of mycelia was severely restricted on the resistant genotype but it was prolific on the sensitive genotype. A bioassay was developed based on germination and extension of the germ tube of U. necator conidia on agar plates. Agar preparations containing desalted crude extracts of grapevine leaves inhibited growth and caused the tips of the hyphae to rupture. The effect was not observed with boiled extracts and was greater with extracts from resistant genotypes. Chitinase and β-1,3-glucanase were purified 760-fold and 46-fold respectively from leaves of Seyval grapevines. The purified enzyme preparations inhibited germ tube growth, with the effect being more prominent in the presence of both enzymes. The results demonstrate that these two enzymes have antifungal activity against U. necator, and are consistent with these pathogenesis-related proteins having a role in defence of grapevines against powdery mildew.

The heteroplasmy of chloroplast DNA (cpDNA) observed in Medicago sativa L., which involves the presence (type B) or absence (type A) of an Xba I restriction site, was examined using closed fragments covering the variable XbaI site from type-A and type-B cpDNA. The 6.2-kb PstI fragment of DNA from type-A cpDNA (−XbaI) and from type-B cpDNA (+XbaI) was cloned into pUC19 plasmids. EcoRI fragments bearing the variable XbaI site from the type-A and type-B 6.2-kb PstI fragments were subcloned into pUC19. DNA sequences of both types of the 696-bp EcoRI fragments were determined and computer-assisted analysis of the sequence data carried out. Type-A cpDNA was found to differ from type-B cpDNA by 1 base, a G to T conversion, which results in a non-recognition site for XbaI in the type-A cpDNA. The sequence difference was in a non-coding region. Cloning and sequencing of the fragments verified the individual identity of the type-A and type-B cpDNA.

The use of hypervariable sequences for DNA typing of plants is focussed on microsatellites and on amplification of regions defined by random (RAPD) or defined (AFLP) primers for PCR analysis of genomes. A hypervariable length of middle repetitive DNA has been isolated from citrus that contains no obvious hypervariable structures. The fingerprinting probe was shown to have an important commercial application in the separation of zygotic from nucellar progeny. A somatic variant of the sequence within one orange tree suggests that somatic variation in hypervariable markers may be a common event.

Seventeen strains of Agrobacterium vitis (formerly classified A. tumefaciens biovar 3) were characterized using part of the T-DNA and virA regions of the Ti plasmid as probes. All strains except one were of the wide host range (WHR) strains and were classified into two groups depending on their ability to utilize octopine or nopaline. These WHR type oncogenic strains had homology with the limited host range type (LHR) virA gen of A. vitis but not with the WHR virA gene of A. tumefaciens.The frequency of T-DNA excision in some Agrobacterium strains was estimated with the plasmid pTMA which mimics T-DNA excision from Ti plasmid DNA. In an A. vitis strain isolated from grapevine, T-DNA excision occurred after co-cultivation with grapevine tissues, but not with acetosyringone. In contrast, in A. tumefaciens, T-DNA excision occurred after co-cultivation with acetosyringone, but not with grapevine tissue.

The role of chloroplast (cp) DNA in plastid and chloroplast function is discussed, particularly in relation to the interaction with nuclear DNA. The evolution of the chloroplast genome and the endosymbiont hypothesis are related to our results and those of others which show the occurrence of cpDNA sequences common to the nuclear and chloroplast genome.

Genetic transformation has been studied in fragmented shoot apex cultures of Vitis vinifera L. following co-cultivation with Agrobacterium tumefaciens. Transgenic shoots of the cultivar Cabernet Sauvignon, tolerant to low levels of kanamycin, have been produced. Proliferation of transgenic cells in the presumptive bud forming area of cultured fragments has been observed using the enzyme activity of β-glucuronidase (GUS) as a marker. The distribution of GUS stained cells both in this tissue and in transgenic shoots, the presence of low copy numbers of the neomycin phosphotransferase II gene in transgenic shoots and the relatively low levels of kanamycin resistance suggest that these shoots contain both transformed and untransformed cells.

Biovar 1 strains ofAgrobacterium tumefaciens have been used to transform a cell suspension culture ofVitis vinifera cv. Cabernet Sauvignon. Cocultivation of cultures withAgrobacterium strains bearing either the cointegrate pGV3850::1103neo, or the binary vector pGA474-68, each gave rise to kanamycin resistant tissue. The stable integration and expression of the neomycin phosphotransferase gene was confirmed by Southern blotting and enzymic assay, respectively.

A 3.4-kbp nuclear (n) DNA sequence has greater than 99% sequence homology to three segments of the chloroplast (cp) genes rps2, psbD/C, and psaA respectively. Each of these cpDNA segments is less than 3 kbp in length and appears to be integrated, at least in part, into several (>5) different sites flanked by unique sequences in the nuclear genome. Some of these sites contain longer homologies to the particular genes, while others are only homologous to smaller parts of the cp genes. Both the cpDNA fragments found in the nuclear genome and their flanking nDNA sequences are invested with short repeated A-T rich sequences but, apart from a hexanucleotide sequence and a palindromic sequence identified near each recombination point, there is no obvious structure that can suggest a mechanism of DNA transfer from the chloroplast to the nucleus in spinach.

Sequences homologous to chloroplast (ct)DNA have been found in nuclear DNA in five species of the Chenopodiaceae, extending the earlier observations of ‘promiscuous’ DNA in Spinacia oleracea (Timmis and Scott 1983). Using the 7.7 kbp spinach ctDNA Pst I fragment as a hybridization probe, several separately located homologies to ctDNA were resolved in the nuclear DNA of Beta vulgaris, Chenopodium quinoa, and Enchylaena tomentosa. In Chenopodium album and Atriplex cinerea the major region of homology was to a nuclear Eco RI fragment (6 kbp) indistinguishable from that in ctDNA. These homologies may therefore involve larger tracts of ctDNA because the same restriction sites are apparently retained in the nucleus. This suggests that in these latter two species there is a contrasting, more homogeneous arrangement of ctDNA transpositions in the nucleus.

A PstI 7.7 kbp fragment from chloroplast (ct) DNA of spinach shows homology to an EcoRI 8.3 kbp fragment of mitochondrial (mt) DNA and in turn, both are homologous to a number of common regions of nuclear (n) DNA. The common area of homology between the chloroplast and mitochondrial fragments is between a KpnI 1.8 segment internal to the PstI sites in the ctDNA and an EcoRI/BamHI 2.9 kbp fragment at one end of the mitochondrial 8.3 kbp fragment. The KpnI 1.8 kbp ctDNA fragment is within a structural gene for the P700 chlorophyll a apoprotein. Further analysis of this KpnI 1.8 kbp fragment confined the homologous region in mtDNA to a ct 0.8 kbp HpaII fragment. These smaller pieces of the organellar genomes share homologies with nuclear DNA as well as displaying unique hybridization sites. The observations reported here demonstrate that there is a common or closely related sequence in all three genetic compartments of the cell.

The discovery of cytoplasmic inheritance in plants at the turn of the century (Correns and Baur as described in Kirk and Tilney-Basset, 1978), culminated in the demonstration of plastid (pt) DNA in the late 1960’s. At the same time there was mounting biochemical and genetic evidence which showed that most of plastid biogenesis and function was controlled by nuclear genes and involved proteins synthesized on cytoplasmic ribosomes (see reviews by Kirck and Tilney-Bassett, 1978; Ellis, 1983). The expression of plastid DNA and the use of plastid ribosomes to synthesize large amounts of particular plastid proteins has only been described in the special case of the photosynthetically competent plastid, the chloroplast (Scott and Possingham, 1980). We wish to distinguish between the general term plastid which describes a family of related plant cell organelles of which the most commonly studied and perhaps the most numerous and important are chloroplasts. In general we will use the name plastid and only use the term chloroplast in specific instances. It appears however that chloroplasts and all other plastids carry an identical subgenome which has been called the plastome. In this article we will briefly describe the interaction of the genetic information from nucleus and plastid that is involved in the formation of chloroplasts and other plastid forms and go on to discuss in more detail the recent observations which indicate that plastids and nuclei share extensive DNA sequence homology (Timmis and Scott, 1983).

The nucleus of eukaryotes contains a complex mixture of DNA sequences (the nuclear genome), whose best understood properties are to control cell division and metabolism. Genomes of plastids and mitochondria are much smaller and are comparable with depleted prokaryotic genomes. These subgenomes only encode a small proportion of the proteins of their respective organelles, the remainder, including most of their own replication systems, being coded in the nucleus, made in the cytoplasm and imported into the organelle. It has been supposed that these subgenomes have unique properties and it is therefore surprising to find that there are many ‘promiscuous’ DNA sequences - sequences which are found in more than one of the three genetic compartments.

Homologies between spinach nuclear (n) DNA and Chloroplast (pt) DNA, have been detected with a clone bank of spinach ptDNA as hybridization probes to restriction fragments of nDNA prepared from purified root nuclei. Every cloned fragment of ptDNA showed homologies to discrete restriction fragments of nDNA, different from those of ptDNA, indicating integration of these homologies into nDNA. While most ptDNA clones were relatively large and probably contained several genes, sequence homologies were also found to the cloned plastid gene for RuBP carboxylase and the β subunit of ptATPase. Many of the homologies in nDNA occur in regions of the genome that are highly methylated and are not digested by the methylation sensitive restriction endonucleases HpaII and MspI. In contrast these enzymes cleave ptDNA into small fragments which allows the nDNA homologies to be distinguished in total root DNA. The sequence homologies observed were not due to contaminating non nuclear sequences as shown by hybridization to mitochondrial (mt) and bacterial DNAs. The total amount of homology to ptDNA in nDNA is equivalent to about five copies of the plastome per haploid nuclear genome. The homologies generally appear to be in individual segments of less than 2 kbp in length, integrated into several different places in the genome.