Prof. R.E.F. Matthews

THE DANIEL McALPINEMEMORIAL LECTURE

Relationships Between Plant Pathologyand Molecular Biology

Professor R.E.F. MatthewsMSc(NZ), PhD ScD(Camb.) FRS FNZIC FRSNZ

Department of Cell Biology,The University of Auckland, New Zealand.

(Delivered to the Opening Session ofThe Fourth International Plant Pathology Congress

The University of Melbourne17 August 1983)

Introduction

Mr. Chairman, Mr. Chancellor, Ladies and Gentlemen,I am greatly honoured to have been asked to give the

McAlpine Memorial Address at the opening of thiscongress.

In spite of living in the Antipodes I have been able toattend all three of the previous congresses held in London,Minneapolis and Munich. However, as I retire in threeyears time this will be the last congress that I will be able toattend. For this reason I am particularly happy to be able toaddress you now. As my subject I have chosen to discussthe developing relationship between two branches ofbiology - plant pathology and molecular biology. At theThird Congress in Munich in 1978 there were two or threepapers on base sequence analysis of viral nucleic acids, asession on viroids, and three papers on the use ofprotoplasts in various aspects of plant pathology.

In the past five years there has been an explosivegrowth of interest and activity in the application of thetechniques and ideas of modern molecular genetics andmolecular biology to problems in plant pathology.Congress Programme here in Melbourne there are twosymposia and several other programmes concerned withthese new approaches. However I doubt that the congressprogramme fully reflects the current activity.

In this lecture I will attempt to give you an overview ofthe current impact of these new techniques and ideas onresearch in plant pathology. By correspondence withcolleagues in various countries I have made a seriousattempt to ensure that the content of my lecture is as up todate as possible. Nevertheless I am sure I will be deficient

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in some respects. I see this situation as being inevitable.The field of plant molecular biology is developing sorapidly that no single person could be au fait with the latestresults from laboratories around the world.

Plant Pathology and Molecular BiologyAs an introduction to the modern era I wish to take a few

moments to review, very briefly, the earlier interactionsbetween plant pathology and molecular biology. Plantpathology is of course much the senior branch of biologyextending back to the early decades of last century. Somewriters consider that molecular biology had its beginningswhen Delbruck, Luria and Hershey formed the phagegroup in 1940. I would place the origin four or five yearsearlier when the first plant viruses were isolated.(a) Virology and Molecular Biology

Stanley crystallised tobacco mosaic virus in 1935 andshowed that it consisted mainly of protein. Rupert Bestmade similar independent findings, here in Australia,about the same time. Bawden and Pirie in 1936 showedthat several plant viruses were nucleo-proteins of theribose type. The apparent dilemma that these self­replicating entities could be crystallised like the proteins ofthe biochemist gave many biologists food for seriousthought. It led to the idea that viruses might make usefulsimple experimental models for understanding thephysical basis of biological reproduction. This was theobjective of the phage group, set up in 1940, that I havealready mentioned.

Since these beginnings, developments in virology andmolecular biology have gone hand in hand. It is worthnoting that Stanley was not a plant pathologist but achemist trained in the study of proteins. Likewise Best wasa chemist. Of the English team Bawden was a plantpathologist while N. W. Pirie was an outstandingbiochemist. I think we can see parallels here with thesituation today. Most of the exciting discoveries at theinterface between plant pathology and molecular biologyare being made either by individuals trained in moleculargenetics and molecular biology or by such individuals incollaboration with establishment plant pathologists.

Between 1940 and 1960 events showed that Delbruckand his colleagues had made an Inspired choice of anexperimental system. Most of the seminal experiments inthe developing field of molecular biology were made usingEscherischia coli and its phages. Nevertheless plantviruses continued to make significant contributions. In the1950s and 1960s some of the more stable plant viruseswhich could be isolated In good yield in a well purifiedstate provided model systems for pioneering theapplication of X-ray crystallographic analysis to the studyof the fine structure of self replicating macro-molecules. In1956 Gierer and Schramm and Fraenkel-Conratdemonstrated for the first time, using tobacco mosaicvirus that RNA molecules could carry genetic information.In 1960 the full sequence of 158 amino acids in the coatprotein of tobacco mosaic virus was established. Thesubsequent study of artificially induced mutations in thisprotein confirmed the universal nature and some otheraspects of the genetic code. However, early interactionsbetween molecular biology and plant pathology were notall highly productive of good science.

In the early post war period the development ofmolecular biology was greatly assisted - in reality madepossible - by the availability of three relatively newinstruments: the Beckmann DU spectrophotometer; theSpinco Ultra-centrifuge and its rotors; and various modelsof the electron microscope. These three instruments

quickly became available to many plant pathologylaboratories especially in the U.S.A. They were frequentlyused by plant pathologists, and especially plant virologiststo produce published papers which in retrospect can beseen as a kind of pseudo-science. For a time use of thesetechniques seemed sufficient to gain editorial acceptance.The only claim to innovation in many papers of this periodwas that they used one or more of these new techniques. Ican make these statements with impunity because I wasan author of at least one such paper myself.

Perhaps the most far reaching contribution made byplant virology to developing molecular biology was atechnical one. In the early 1950s M.K. Brakke developedsucrose density gradient centrifugation as a method forpurifying viruses. This technique was rapidly adopted andadapted by virologists and molecular biologists of allpersuasions. It has been, and still is, a key technique in thearmoury of molecular biology.

In the 1950s methods for growing and assayingvertebrate viruses in cultured cells were developed. Thisled to a rapid increase in knowledge about the structureand replication of these viruses. Plant viruses fell into thirdplace behind bacterial and vertebrate viruses as modelsystems for study by molecular biologists. Only in the lastfew years have they begun to catch up.

There is one notable exception to the above statement.About 12 years ago viroids were discovered by plantvirologists, and these have turned out to be a fascinatinggroup of disease-inducing agents - a challenge and anenigma for both plant pathologists and molecularbiologists. Viroids are the smallest Known infectious self­replicating pathogenic agents. Their structure has beenfully determined. They consist of a single-strandedcovalently closed circular RNA molecule. The size variesfrom 250-600 nucleotides. Their mechanism of replicationis partly understood, but the way in which they causedisease is quite unknown. An intriguing feature is the verysmall change in nucleotide sequence needed to produce amarked change in disease expression. For example thedifference between strains of potato spindle tuber viroidcausing severe or mild disease consists of a change ofbases in only three sites in the molecule. As will bereported at this congress by T.O. Diener, the constructionof deletion and substitution mutants of this viroid is nowpossible. These should permit the identification of regionsof the molecule involved in specific functions including theinduction of disease.

Several indigenous viruses in Australia have been foundto contain a small viroid-like RNA molecule as well as alarger linear RNA. Further study of these intriguing virusesmay throw some further light on the origins and nature ofviroids.

(b) Molecular Biology and Other Aspects of PlantPathology

So far I have been referring only to plant viruses andrelated agents. What of the other branches of plantpathology - mycology, bacteriology and nematology. Asfar as I am aware there has been very little interactionbetween these areas and molecular biology until therecent developments in the use of protoplasts, DNAprobes, gene transfer, etc. which I will now consider.

Many applications of these new technologies areemerging, such as: refined methods for disease diagnosis,methods for determining relationships between strains ofpathogens; for studies on the mechanism of inducedresistance and the hypersensitive response; and the use ofmolecular markers in standard plant breeding. However

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their most important potential use, from the plant diseasepoint of view, is of course the development, by novelmeans of host varieties resistant to a particular agent.

(c) New Technologies in Plant PathologyI will now discuss some of these applications of the new

technologies to problems in plant pathology. In the timeavailable my comments must, of necessity be brief.

(i) Diagnosis of DiseaseTwo of the newer methods are being developed as

diagnostic tools, especially for diseases caused by virusesand viroids. There are nucleic acid and hybridizations andmonoclonal antibodies. Serological diagnosis cannot beused for viroids because there is no associated protein.Furthermore, symptom development following inoculationmay take up to two years. Thus nucleic acid hybridizationusing a labelled C-DNA probe is proving particularly usefulas a diagnostic tool for this class of agent.

So far, 32p has been used to label most viral and viroidDNA probes. However, probes labelled with a non­radioactive chemical, biotin, have been developed, andrecently a rapid and sensitive method for visualisingbiotin-labelled probes has been described. The procedurewill detect target polynucleotide sequences in the 1-10 pgrange. This should greatly facilitate routine diagnostictesting in laboratories with minimal facilities.

A wide variety of serological procedures usingpolyclonal antisera have been used for a long time asdiagnostic tests, with variations on the 'Elisa' procedureproving very popular in recent years. Monoclonalantibodies will offer the possibility of very high specificityin diagnostic tests - perhaps too high for some purposes.

(ii) Relationships between strains of pathogensThis problem is related to the question of diagnosis.

Delineation of strain relationships may be greatly assistedboth by nucleic acid hybridization and monoclonalantibody techniques - especially for bacterial and viralpathogens.

Restriction enzyme mapping of viruses with double­stranded DNA genomes is a useful alternative tohybridization. Another alternative, applicable to RNAviruses is fingerprinting of enzyme digests of labelled RNAby two dimensional gel electrophoresis.

(iii) Mechanisms of disease ResistanceThere has been widespread interest for many years in

the mechanisms of disease resistance in plants, but thesemechanisms have proved difficult to pin down byconventional physiological and biochemical methods. Thetechniques made available by the new DNA technology areallowing fresh and constructive approaches to be made tothe problems of disease resistance. I will give you oneexample. It has been thought for some time that theaccumulation and release of phytoalexins in plant cells inresponse to infection by fungi or bacteria is an importantfactor in resistance to some pathogens. The enzymechalcone synthase is the first enzyme on a branch pathwayleading to the biosynthesis of phytoalexins fromphenylalanine. In experiments to be discussed at thiscongress, C.J. Lamb has used cloned C-DNA probes tochalcone synthase messenger RNA to show that there is avery clear difference in the expression of this gene incultivars of Phaseolus vulgaris susceptible or resistant to aspecies of Colletotrichum. In a resistant cultivar there is arapid induction of chalcone synthase messenger RNAwhich is detectable within 20 minutes, and which islocalised at the initial site of infection. In a susceptiblecultivar, there is some induction of the enzyme but it

occurs much later and at sites distant from the initial site ofinfection.

Lamb has shown that in elicitor-treated bean cellcultures the increased amounts of messenger RNA involvede novo m-RNA synthesis. Thus, the induction ofphytoalexin synthesis in a resistant cultivar reflects a veryrapid and extensive switch in gene expression on the partof the host plant.

This study opens the way to an examination of how theelicitors of phytoalexin synthesis found in fungal cell wallsand elsewhere, actually act on the host genome so rapidlyafter the infection process begins.

The molecular biology of resistance to obligate fungalparasites such as the rusts may be much more difficult tounravel. As far as I am aware, no-one has yet isolated theproduct of a gene for resistance to an obligate fungalparasite.(iv) Molecular Biology and Generating Disease Resistant

CultivarsPlant pathologists and plant breeders have recognised

for a very long time that the most effective form of diseasecontrol is to find or develop disease-resistant cultivars. Theimportance of this form of disease control has beenstrongly reinforced in recent decades with the realisationof the potential ecological hazards involved in thewidespread use of many forms of chemical control. Thusthe most active interactions between molecular biologyand plant pathology at the present time relate to the questfor novel means of generating disease-resistant cultivars. Iwill consider this topic for the remainder of the lecture.

The Quest for Disease Resistant CultivarsThere are two main groups of techniques to be

considered - the use of protoplasts and tissue culturegenerally, and - the search for vectors to introduceforeign DNA into plants.

1. Tissue Culture and protoplastsFor clonally propagated plants there are several aspects

of tissue culture that are of indirect value in plant breedingprogrammes. Many virus-infected species can be freed ofvirus infection under appropriate conditions of tissueculture. This in itself is often of practical value, but it alsomeans that varietal material can be distributed morewidely from country to country through quarantine. Ofmore direct interest is the genetic variation revealed inplants regenerated from cells or tissue grown in culture.The potential value of this somaclonal variation firstbecame apparent in work with sugar cane some 15 yearsago. However, its potential has recently received wideattention particularly as a result of work on potatoes in J.F.Shepard's laboratory. Clonal populations regeneratedfrom single leaf cell protoplasts of the cultivar 'RussettBurbank" showed a high frequency of variation in variouscharacters including enhanced resistance to early blight(Alternaria solani) and late blight (Phytophthora infestans).

Heritable variation arising in culture is not confined tovegetatively propagated species. For example, a widerange of such variation has been found in wheat byScowcroft and his colleagues in Canberra. Variation hasbeen obtained not only from protoplast culture, but alsofrom callus tissue, immature pollen, ovules, and immatureembryos.

Variation seen in the first generation of somaclonalplants may have either an epigenetic or a genetic basis. Tobe useful in a breeding programme variation must beshown to survive a meiosis. It is possible to treat thecultured cells or tissue with known physical or chemicalmutagens to attempt to increase the range of variation.

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This approach is being followed in several laboratories.For the relatively few diseases for which a toxin has

been isolated and characterised it may be possible toscreen large numbers of protoplasts or calli in vitro. Forexample maize plants resistant to Drechslera maydis havebeen regenerated from callus grown and regenerated inthe presence of the growth inhibiting toxin from thefungus. In this kind of screening programme it is importantto use a chemically defined toxin that is known to beinvolved either in pathogenicity or in virulence. Crudeculture fitrates are unlikely to be much use.

Selection for resistance at the tissue culture stage will,of course not be possible for the many plant diseaseswhere there is a complex host-pathogen interaction, forexample rusts and mildews. For these, selection must beapplied to the regenerated plants.

Somaclonal variation is superimposed directly on thequalities of the cultivar from which the tissue was obtainedwithout the introduction of a vast array of new alleles as ina sexual cross. In spite of this, the full evaluation in the fieldunder various conditions of any interesting plantsregenerated from tissue culture will take several years.

Much of modern plant breeding aims to introducedisease resistance genes from distantly related species.Tissue culture offers two procedures for extending therange of such crosses. First, fusion of protoplasts fromdifferent species. So far this has been confined mainly tothe Solanaceae. Second, in-vitro fertilisation followed byculture of the egg or ovule.

Tissue culture methods promise to make a significantcontribution to the development of new disease resistancecultivars. Nevertheless, there are many difficulties. Thecontrol of regeneration of viable plants from tissue cultureis not understood in any species, and there is markedvariation between species in the ease of regeneration.Selection in tissue culture for whole plant characters thatare polygenically controlled is not possible. Finally theprocedures are all based on the chance emergence of auseful variant.

2. Vectors for introducing foreign genes into plantsThe molecular biological approaches I now wish to

discuss offer the possibility of introducing a single definedgene from any source, into a plant. In favourablecircumstances the techniques of DNA manipulation nowallow the structural gene for a particular protein to beisolated from a gene library of the organism concerned.The gene may then be joined to any required controllingDNA elements and amplified by replication in Escherichiacoli.

In principle the isolated gene may then be introducedinto a new organism, in our situation a crop plant. Again inprinciple this procedure has substantial advantages overconventional breeding: Firstly, it avoids extensive back­crossing. Secondly, it is highly directed. Thirdly, itfacilitates the introduction of entirely novel geneticproperties. However, just how to introduce the isolatedgene is a major question.

Gene VectorsI will now survey briefly the kinds of potential vector that

are under study in many laboratories for the introductionof foreign DNA into plants.

For an effective plant gene vector system severalfeatures are required: Firstly, it must maintain and transferthe DNA through successive cell divisions. Secondly, theintroduced DNA must be correctly expressed in the cell inorder to alter the cell's phenotype. Thirdly, any vector

based on a plant pathogen must be so attenuated as toremain capable of infection while causing no significantdisease. Fourthly, for many crop plants the introducedgene should be stably maintained through meiosis, so thatit could be used in a plant breeding programme.

Three kinds of vector system are being activelyinvestigated: (i) those based on 1; plasmids; (ii) thosebased on plant viruses; and (iii) those based on the directintroduction of DNA into a cell.1. TI plasmids as VectorsThe ability of Agrobacterium tumefaciens to causetumours in many dicolyledonous plants is due to thepresence of a large plasmid - the tumour inducing or TIplasmid. Tumours are caused by the stable integration ofpart of the TI plasmid known as the T-DNA into thenuclear DNA of the plant cell. Although the T-DNA is foundin bacteria it is functionally active in plant cells, producingtypical eukaryote m-RNA's. Seven of these have beenrecognised. Expression of T-DNA causes a hormone in­balance leading to tumour formation. The T-DNA alsocodes for enzymes which cause the transformed plantcells to produce unusual amino acid derivatives called'opines' which are used by the Agroacterium as a source ofcarbon and nitrogen. Thus the Tr plasmid functions as anatural plant vector for DNA.

Standard DNA manipulation techniques have been usedin several laboratories to insert foreign DNA into the T­region. The Agrobacterium then introduced theengineered DNA into the plant cell. In early experimentsvarious laboratories failed to achieve expression of foreigngenes inserted into the 'T' sequences. In addition it hasproved difficult to regenerate fertile plants from tumourtissue. However, Schell's group have reported amorphologically normal plant that spontaneouslyregenerated from tumour tissue. The plant stably main­tained and expressed the gene for octopine synthase.

Furthermore, this enzyme activity was inherited as adominant Mendelian marker. The T-DNA had suffereddeletions in the tumour controlling genes. Recently threegroups have constructed a chimeric T-DNA that contains abacterial gene which is expressed in plant cells. In all threelaboratories the coding region was excised from aT-DNA.A bacterial gene for kanamycin resistance was splicedbetween the T-DNA regulatory regions. This chimeric DNAwas then introduced into a TI plasmid. Plant cellstransformed by the plasmid were resistant to kanamycin.

This work appears to open the door to the transfer ofvirtually any foreign gene into a plant cell. Antibioticresistance provides a selectable marker for integration;while disabling of the controlling genes may allow forregeneration of plants in suitable species. Perhaps theone major limitation of T-DNA as a vector is thatAgrobacterium does not infect monocotyledons, whichinclude many of our most important food plants. However,ways may be found to extend the host range of a vectordisabled with respect to gall formation.

Besides the TI plasmids some other DNA moleculeswith certain plasmid-like properties are known in plants,and these might be potential gene vectors. For examplethe mitochondria of maize may contain at least six differentextrachromosomal forms of DNA. One of these is like asmall cryptic self-replicating plasmid. However, they carryno known markers; and if they replicate only within themitochondrion, there may be substantial difficulties inreturning an engineered molecule to this organelle. Thus,although much remains to be learned about the possibleuses of plasmids as plant gene vectors, substantialprogress has been made in the past two years.

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2. Plant Viruses as Gene VectorsSeveral laboratories have been actively exploring the

possibility of adding a foreign gene to a viral genome sothat the gene would be introduced into a plant cell duringthe infection process, and perhaps be expressed there.Such a vector system would differ in an important wayfrom the Agrobacterium plasmid vector.

Viruses can move from cell to cell through the plant.Thus a viral vector would be able to introduce a gene intogrowing intact plants thus avoiding the problemsassociated with regenerating plants from single cells. Mostexperimental work on virus vectors has concentrated oncauliflower mosaic virus. This has a double-stranded DNAgenome, making it directly amenable to genemanipulation techniques. The genome of about 8,000 basepairs has been fully sequenced. It is housed in anicosahedral particle about 50 mm in diameter.

A key experiment was reported from Howell's laboratoryin 1980. They showed that copies of the viral DNApropagated in E.coli using a plasmid vector wereinfectious when inoculated onto host plants. This openedthe way for performing recombinant DNA manipulationson the viral genome in E.coli and for testing their effects inplants.

There are several sites where foreign DNA might beinserted into the viral DNA without destroying infectivity.There is one large and one small intergenic region. Inaddition two non-essential coding regions have beenidentified. Small insertions of bacterial DNA at these non­essential sites were successfully propagated through acycle of infection in the plant. However, these experimentsrevealed two serious problems. First, there appears to bea stringent limitation on the amount of extra DNA whichcan be inserted without causing deletions. This is about250 base pairs. However when insert stability is betterunderstood it may be possible to delete enough non­essential DNA to allow for the insertion of about 1,000 basepairs of foreign DNA. This would be enough to code for anaverage size protein. The second limitation is perhapsmore serious. During several cycles of propagation of anengineered virus in plants the inserted DNA tends to belost through recombination.

Further factors limiting the potential use of this virus arethat its host range is confined to the Cruciferae; and that itis not seed transmitted. Thus, at this stage it seemsunlikely that cauliflower mosaic virus will become aneconomically important vector. Its greatest use will be as atool for studying gene expression. Already, two cauliflowermosaic virus promotors have been identified. These directhigh rates of transcription when copies of the virus areintegrated into plant chromosomes using the T, plasmid.Thus the virus may prove to be a useful source ofpromotors for the expression of foreign genes in plants.

One other group of DNA plant viruses is known - 'TheGeminiviruses'. Several laboratories are exploring theirpotential as gene vectors. However, they have some poorfeatures. Most are confined to the phloem tissue, and thevery small size of the virus particles suggests that thepackaging problem may be at least as severe as with theCaulimoviruses.

For a time it was thought that RNA viruses might havelittle prospect of being developed as gene vectors.However, it has been established for several RNA virusesinfecting bacteria or mammals, that a full length DNA copyof the RNA is infectious for appropriate cells in culture.These findings opened the way for making the RNAgenomes amenable to the various manipulations that canbe carried out in vitro on double-stranded DNA.

At present, various laboratories are exploring the use ofplant viruses with RNA genomes. Many plant viruses havetheir genomes divided into two or three separate pieces ofRNA. Some of these multipartite viruses are particularlyattractive as potential vectors. For example tobacco rattlevirus has a large and a small RNA housed in two separaterod-shaped particles. The small RNA contains the coatprotein gene, which is not essential for the replication ofthe larger RNA as a naked RNA. The possibility thereforeexists of introducing foreign RNA (via a dsDNAintermediate) with either the long or the short RNA andachieving replication of the engineered molecule.Furthermore there would be no packaging problem with anaked RNA.

Some isolates of several RNA plant viruses containsatellite RNAs which are not essential for replication of thevirus. They are templates for their own replication, butrequire functions of the helper virus for that replication. Itappears possible that additional genetic material could beinserted into the satellite RNA without affecting replicationof the virus.

Viroids have certain features which make themattractive as potential vectors. They move systemicallythrough an infected plant; they are sap-transmissible;some are transmitted through the seed; they appear toreplicate in the nucleus; and they infect a wide range ofplants including some important tropical crops.

The entire genome of one viroid has been cloned insuch a way that it can be clearly excised from the plasmid.This DNA copy is infectious. Thus the way is open fortesting the effects of insertion of foreign DNA into theviroid.

A significant difficulty with all the viruses and the viroidsdiscussed so far is that they normally cause disease intheir host plants. For economically effective use asvectors, the viral gene or genes responsible for diseasedevelopment would need to be identified and disabled.

It would not really be sufficient to use a symptomlessstrain as a vector. This is because a double infection in thefield with an unrelated virus would have a good chance ofcausing very severe disease.

With respect to this problem of gene vector virusescausing disease, a recently described group of agentsfound in such plants as beet and carnations will be worthinvestigation. These are the so-called cryptic viruses. Theyhave small isometric particles, occur in low concentrationin the plant, and usually cause no symptoms at all. Theyare not transmissible by mechanical inoculation or even bygrafting, but they have a high rate of seed transmission ­a very useful feature for a potential gene vector.

To summarise the situation with plant viruses aspotential gene vectors, on the information available so farthey do not look as promising as the plasmid vector.However, in spite of the progress made with T I plasmidvectors it may still be well worthwhile to pursue the virusvector possibility, especially for perennial crops. Imaginesome time in the future when the owner of a largeestablished apple orchard has the option of introducingresistance to a fungal disease into his orchard either byinoculating his existing trees with an engineered virus, orby replacing his existing trees with new ones givenresistance via a plasmid. I think he would choose the virus.

3. Direct DNA-mediated gene transferIt is possible to insert DNA into plant cells directly

without using a biological vector. Such DNA may be takenup and integrated into the plant genome. There are twopossible strategies to overcome the barrier to the entry of

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DNA presented by the plant cell wall: - remove the wall togive protoplasts, or micro inject the DNA into an intact cell.Some nucleic acids can be taken up by freshly isolatedprotoptasts, This uptake can lead to a high proportion ofthe protoplasts becoming infected. Thus nucleic acids canbe taken up by protoplasts without loss of biological ac­tivity.

More interesting, protoplasts of at least two species,petunia and tobacco have been transformed byAgrobacterium TI plasmid DNA. Transformed colonieswere identified by their ability to grow in the absence ofexogenous hormones.

The other alternative for transforming cells directly withDNA - micro injection into the nucleus has beensuccessfully applied to some mammalian systems.Although only a small number of cells can be injected, thefrequency of gene transfer is high enough - up to about20% - that no selection for transformation is necessary.Interest in the mammalian experiments has promptedseveral groups to investigate micro-injection for plantcells. This approach could be combined with the use oftransposable elements linked to the gene of interest tofacilitate integration of the gene into the host chromo­some. If successful transformation could be achieved inpollen or egg cells or in the embryo, new genes could beintroduced directly into a crop plant. The need forregeneration of whole plants from protoplasts or callus tis­sue might be bipassed.

To summarise the present situation with respect to plantgene vectors: Present indications are that modified T Iplasmids, rather than viruses will provide the firstsuccessful vector for a useful gene.

Concluding RemarksWe have seen that regeneration of a wnole plant

expressing a gene from tumour DNA has been achieved,and that such expression was inherited in a Mendelianfashion. However, there remain further requirements yetto be faced. To be useful, the introduced gene must beexpressed at an appropriate site in a co-ordinated fashion.For example a gene for resistance to a leaf spot funguswould be little use if it was expressed only in the roots. Afurther very major problem relates to all attempts at genetransfer - and not only genes for disease resistance. Wefirst have to be able to lay our hands on the gene we want.

There is no general method available for identifying andisolating the gene or genes responsible for a particularcharacter from a gene library of the organism containingthe gene. At present, we have to be able to isolate themRNA or the protein product before the gene itself can beisolated.

I expect that there are many plant breeders in thisaudience and around the world who are saying "Look whatconventional plant breeding has done for the crops ofmankind. What has molecular biology contributedcontributed - almost zero."

I will answer that point and conclude this address byquoting from Gunther Stent, a well-known molecularbloloqtst, In 1969 Stent wrote a book sub-titled "The Com­ing of the Golden Age: A View of the End of Progress'. Thefirst section of the book is entitled "The Rise and Fall ofMolecular Genetics". In the prologue he says, and I quote:"I have devoted the first four chapters to the field in which Iam a professional, namely molecular genetics. I willdescribe the history of my field in order to show that its riseand fall is but a paradigm of the history of creative activityin general". The explosive growth of technology and ideas

in molecular genetics over the past few years show thatStent's pessimism was quite unjustified - or at the least,very premature. I am an optimist, and I believe the nextdecade will see the new ideas and techniques of molecularbiology and molecular genetics successfully applied forthe benefit of man in the control of plant diseases and inmany other fields as well.

rapid colonization by P. cinnamomi, we also had observedrenewed and rapid invasion of inoculated jarrah coppicestems after unusually heavy rainfall at Dwellingup duringJanuary 1982 (January rainfall, 1982 was 237 mm; average13 mm).

RESEARCH NOTES 36 f-

The Relationship Between Bark Moistureand Invasion of Eucalyptus marginata byPhytophthora clnnamoml

32t-

28 f-

Fig. 1. Bar diagram showing mean lesion lengths for coppice and'R' site sapling stems at bark moisture increments of 3%. Pointsrepresent length of individual lesions recorded within each barkmoisture increment.

K24f­s:OJc

j"r16L

12~81-

In order to examine the relationship between barkmoisture and lesion development, three different sets oftrees were inoculated during March 1983 near Jarrahdale,Western Australia. Fifteen jarrah saplings near apermanent creek ('R' site vegetation type, Havel (5)classification), 15 similar saplings on a drier upland site('S' type) were inoculated. Inoculation was carried out by amethod similar to that described by Tippett et et. (12).Since the mean maximum day temperatures for theduration of the experiment was near 28°C, (Perthrecordings) temperature would not have limited thegrowth of P. cinnamomi to any great extent (10). At thetime of harvest, 19 days post-inoculation, relative barkmoisture levels were determined using the method of Bier(3) and lesions were measured and mapped.

Bark moisture was shown to be an important factoraffecting lesion development. Of the saplings growing nearthe creek, the mean relative bark moisture level was84.8%. Mean lesion length was 12.2cm. Bark moisturelevels in these stems varied between 74% and 90%. There

Relative bark moisture (%)

94I

91I

88

I

85I

82

I

79I

76I

73

4f-

Joanna T. Tippett and T.C. HillForests Department,

Hayman Road, Como, W.A. 6152

Lesion development caused by the invasion ofsecondary phloem of Eucalyptus marginata Sm. byPhytophthora cinnamomi Rands has been followed overthe past two years. Coppice stems and large roots wereinoculated during spring, summer and autumn and typicallesions have been described (12). It has been shown thatjarrah can express resistance during most of the year; thefungus was arrested and lesions were bound by necro­phylactic ('wound') periderm in at least 60% of theinoculated coppice stems and most of the inoculatedroots. Initial establishment of the fungus in the phloem wasmost rapid during early summer (Shea and Deegan,unpublished) when day temperatures were already nearoptimal for fungal growth (13). Inoculations made duringFebruary and March generally resulted in shorter lesions.When lesions were left to develop for at least 12 months,stem dissections indicated that fungal activity had beenintermittent; under certain circumstances the fungusrenewed advance from lesions which had beentemporarily confined (12). The fungus girdled stems wheninitial establishment was rapid or when it 'broke-out' fromexisting lesions. Marks (7) have made similar observationsof lesion development in susceptible Victorian eucalypts.

Rands (9) in his original description of stipe cankercaused by P. cinnamomi in cinnamon trees also notedintermittent growth of the fungus. Lesions had a patternwhich reflected 'periodicity in canker spread'. Randsinvestigated the reasons for the intermittent growth of thefungus, the periodicity being of an apparently shorterinterval than we have observed in jarrah. He concludedthat 'rhythmic spread of the fungus was probablycorrelated with a habitual periodicity in the physiologicalprocesses in the tree'. His observations were neverexplained. We now suggest that fluctuations in barkmoisture influence fungal activity.

Although temperature has been acknowledged ashaving a major influence on fungal growth otherenvironmental factors were sought which adverselyaffected jarrah's ability to resist invasion. Water stressaffects the rate of some defence reactions in trees (8) andprevious work has shown that Populus and French prunewere most susceptible to canker organisms (e.g.Crypodiaporthe sa/ice//a, Fusarium lateritum, Cytosporaleucostoma) when bark moisture levels were at theirlowest (2, 3). Despite the possibility that water stress couldcause a decrease in resistance, or predispose jarrah to

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