Acta Bot. Neerl. June 161-171 40(2), 1991, p.

Meetings of the Royal Botanical Society of

The

MEETING OF THE SECTION FOR PLANT MORPHOLOGY,

ANATOMY AND CYTOLOGY ON 26 OCTOBER 1990

al. Anatomy and Pyrolysis Mass Spectrometry unknown) (van der Heijden et 1990, Proceedings Peat 90, (ed. Sopo, R.), 148-163). Anatomical ofPeatified PlantTissues pp.

changes were observed, however: the pith, as well E. van der Heijden and J.J. Boon. Unit for Mass cortex as the are easily decomposed, although the Spectrometry ofMacromolecular Systems, FOM cell corners are often retained. In areas where Institute for Atomic and Molecular Physics, decomposition is severe, high fungal activity is also Kruislaan 407, 1098 SJ Amsterdam and Unit for observed. Molecular Paleobotany, Hugo de Vries-Laboratory,

University ofAmsterdam,Kruislaan 318, 1098 SM

Amsterdam,The Netherlands Effect ofGrowth Rate and Age on Wood

Aselection ofpeatifiedplant tissues, handpickedfrom Structure of East-Liaoning Oak

and a Dutch peat deposit, were anatomically mass S. Zhang and Y. Zhong. Rijksherbarium, P. O. Box

spectrometrically characterized and compared with 9514,2300 RA Leiden, The Netherlands

native tissues. In this way, insight is obtained in

the chemical and anatomical selectivity of the The effect of growthrate on wood structure of East-

in Oak decompositionprocess peat deposits. Liaoning (Quercus liaotungensis Koidz.) was

A selective removal of polysaccharides and a studied, and compared with the effect of age (ring

trees structural modification of the lignin macromolecule number from the pith). Five dominant were

are observed in both peatified Calluna stemwood and collected from Mt. Zhongtiao, North China. The

Ericaceous rootwood. These chemical changes coin- studies revealed that age is a decisive factor controll-

cide with severe anatomical changes, although the ing wood structure, while the effect of growth rate is

state of preservation between different wood forming less important.

tissues is rather variable: in peatified Calluna fibres, The effect ofage on anatomical characters shows

the combined layer is whereas the S optimum curves which differ from character to S,/S2 gone 3

layer and compound middle lamella are preserved. character. For fibre proportion, the effect shows a

Sporadically, fibres with a higher or lower grade of parabolic curve, with an optimum age from 25 to 35

observed. The wood show- for maximum size. decompositionare vessels, years

ing swollen and gelified cell walls, are better preserved The effect of growth rate varies not only with

than the surrounding fibres. A uniform thinning of anatomical characters but with the position within a

the cell wall is observed in the rootwood fibres, which growthring (earlywood, latewood or the whole ring),

often better in the of the width and The effect of are preserved centre root. ring range age. growthrate on

Decomposition resistent wood vessels, as found in sizes of wood elements (fibre length & diameter and

Calluna stemwood, are not observed in the wood of vessel diameter) is not significant. However, growth

the root. rate shows a greater effect on tissue proportions.

The bark of the Calluna stem is highly resistent Growth rate does not influence characters of either

to decomposition. The periderm, which is the most earlywood or latewood significantly, but it shows a

resistent tissue, is not modified. Minor anatomical greater effect on characters ofthe whole growthring.

modifications such cell wall and Within of width than as thickening gelifica- a narrower range ring (less

tion, are observed in the phloem parenchyma and about 1 -5 mm), with increasing ring width (or growth

cambium. Mass spectrometric analysis of peatified rate), fibre proportionincreases rapidly, while vessel

not bark reveals that polysaccharides are decomposed and parenchyma proportion (including vasicentric

during peatification. tracheids) decrease markedly, then more slowly, and

The stems of Sphagnum are very resistent to finally they remain more or less constant when ring

The of width is than decomposition. mass spectrum peatified beyond a specific range (wider c.

Sphagnum is still characterized by intense mass 3 0 mm). Growth rate shows little effect on the struc-

peaks of polysaccharides, which points to an excel- ture of juvenile wood, but a significant effect was

lent preservation of these compounds (mechanism found in mature wood.

161 162 MEETINGS

of cell differentiation cell Bi- and Unitegmy in Impatiens express some type or even degeneration. F.D. Boesewinkel. Hugo de Vries-laboratory, It be hypothesized that, perhaps as a result of University of Amsterdam,Kruislaan 318, 1098 SM may

unfavourable small occur in a Amsterdam, The Netherlands conditions, rings tran- sition where function available stage, they as a readily Within the the transition of bi- to genus Impatiens F-actin pool. unitegmy occurs. Impatiens glanduliferais one of the bitegmic, I. noli-tangere one of the intermediate and Deposition and Reorientationof Cellulose I. congolensis one of the unitegmic . The Microfibrils in Cells of unitegmic condition arises by the upward growth of Elongating Stylar Petunia subdermal cells under the dermal outer integument, hybrida togetherwith the arrested growth and shifting of this A.M.C. Wolters-Artsand M.M.A. Sassen. integument along the inner integument to a more Department ofExperimental Botany, University of apical position. In the unitegmic species the earlier Nijmegen, Toernooiveld,NL-6525 ED Nijmegen,

does and the The Netherlands outer integument not develop anymore single integument consists mainly of the former inner According to the Multinet-Growth-Hypothesis integument and the subdermal cells which grow far (MGH) (Roelofsen & Houwink, Acla Bot. Neerl. upwards. The endothelium becomes the inner layer of 1953, 2: 218-225), originally transversely deposited the singleintegument. microfibrils become gradually re-oriented towards This isafourthwayfor ovules to become unitegmic. In more axial orientations during cell expansion. order Other in which ovules become ways may unitegmic to establish the extent ofmicrofibril reorientation, we are: quantitatively studied microfibril deposition and the (a) reduction ofthe outerintegument, texture during elongation in stylar parenchyma and (b) fusion oftwo dermal integuments,and transmitting tissue cells of Petunia hybrida. (c) integumentary shifting of the inner integu- of From the inner surfaces very young cells in styles ment by a subdermal outer integument. smaller than 0T cm, the following sequence in depo- The study ofovules in taxa in which the change over

sition was inferred: microflbrils were deposited in of bi- to unitegmy takes place, gives information on alternating S- and Z-helical orientations. First nearly the evolution of the unitegmic ovule. axial orientations, followed by oblique and nearly

transverse orientations. Together with the increasing Actin Rings are a Regular Constituentof pitch, the density increased. Only nearly-transverse PlantCells S- and Z-helical orientations were deposited during

H.M.P. of Kengen. Department Experimental elongation. The wall maintained its thickness and

Toernooiveld Botany, University of Nijmegen, 1, initial texture.

6525 ED The Netherlands Nijmegen, In cells the from axial young stylar sequence to

transverse microfibril orientations reflects the way in Rhodamine-labelled phalloidin was used as a probe

which microfibrils are deposited. Duringelongationa to study F-actin distribution in suspension cells of continuous deposition of nearly transversely oriented Nicotiana and Tagetes, and pollen tubes ofLilium. In microfibrils takes place. However, no perceptible addition, we also studied elongating suspension cells, changes are observed in the total texture during cell protoplasts from the suspension cells and subproto-

expansion. The initial texture is maintained asa result plasts from pollen tubes in Nicotiana. Actin rings were ofpassive re-orientation, asdescribed by Roelofsen & present in all cells studied. In each preparationring- Houwink. The extent of passive re-orientation is in like structures were observed in about 5% of the cells with the theoretical calculations of Preston which occurred after fixation with paraformaldehyde agreement (Planla 1982, 155: 356-363) and shows that the total in PIPES-buffer (Traas, 1984, Protoplasma. 119, reorientation is limited to 40° in transmitting tissue buffer 212-218), as well as after extraction in PIPES cells and 59° in stylar parenchyma cells. with non-ionic detergents (Traas el al. 1987, J. Cell

Biol.. 105: 387-395),

Simultaneous visualization offluorescent and phase The Apertural System in Nephelieae Pollen

contrast images revealed no perceptive association (Sapindaceae): Form, Function, and with organelles.The ofrings duringorganelle presence Evolution division (Hasezawa el al. 1988, Proloplasma, 146: R.W.J.M. Van der Ham. Rijksherbarium/Hortus 61-63) could not be confirmed. Botanicus, University of Leiden, P.O. Box 9514,2300 As the rings are present in a limited number ofcells RA Leiden, The Netherlands of the (about 5%), they may express a specific stage

The cell cycle. At present, their occurrence in synchron- Nephelieae represent a rather eurypalynous tribe

ized cell cultures is studied. Alternatively, they may ofthe family Sapindaceae, both with regard to exine MEETINGS 163

architecture and apertural system. The apertural 1989, New Phytol. Ill, 323-358). These distributions system is tri-aperturate. Three types occur in the 12 were observed in pollen tubes afterchemical fixations, genera of the Nephelieae: colporate (ectoapertures which may have caused distortions in cytoplasmic free; in 11 genera), parasyncolporate (ectoaper- organization (Mersey & McCulley 1979, J. Microsc. tures connected; in Alectryon), and brevicolporate 114, 49-76). Therefore, we have used cryofixed and

(ectoaperturesshort; in Pometia). The genusAlectryon freeze substituted pollen tubes of Nicotiana tabacum cv shows a continuous range from colporateto parasyn- Samsun. Ultrastructure and distribution oforganelles colporate. These two types occur together in several in the growingtip were studied on longitudinalserial other sapindaceous tribes. sections. The results showed an accumulation of

It is widely accepted that within the angiosperms, vesicles in the extreme tip. Also mitochondria, colporate is more primitive than parasyncolporate, dictyosomes, smooth and rough endoplasmic but this is questionable with regard to the Nephelieae reticulum showed an increased density in distinct and most other tribes oftheSapindaceae.On account of zones (mostly between 10 and 40 pm) behind the tip. macromorphological data and a functional interpret- In this zone also many, noncortical, microtubules ation of both it is which be involved in apertural systems, hypothesized occur, may maintainingorganelle that the primitive colporate condition ofangiosperm distribution. These results largely confirm the earlier

persists in the subfamily Dodonaeoideae but data on organelle distribution (Steer & Steer 1989). pollen , that parasyncolporate is the primitive state in the We also observed vesicles and dictyosomes seemingly other subfamily, the Sapindoideae. The colporate attached to filaments, putatively F-actin. At the

in the latter is considered be derived it is between 5 and 20 behind the state to as a plasmamembrane pm tip, reversal. Alectryon pollen provides some ontogenetic an accumulation of coated pits (up to 10 coated pits evidence. Fossil evidence can hardly be used: fos- per micrometre membrane per section) occurs. These sils of the parasyncolporate type are well known coated pits are probably involved in membrane

(Cupanieidites), but fossil colporate Sapindaceae retrieval to compensate for the excess of membrane

is difficult from other in the pollen to distinguish many incorporation growing tip.

families.

The evolution from the parasyncolporate to the PollenTube Interaction with the Ovule colporate condition could have occurred frequently, During the Progamic Phase in Gasteria possibly under the influence of the worldwide aridifi- cation and cooling in the course of the Tertiary, verrucosa (Mill.) H. Duval

as the two apertural types show quite different M.A.W. Franssen-Verheyen and M.T.M. Willemse.

harmomegathic systems. A colporate pollen grain DepartmentofPlant Cytology and Morphology,

accommodates volume reduction (due to water loss AgriculturalUniversity, Arboretumlaan 4,6703 BD

after anther dehiscence) by invaginating especially Wageningen,The Netherlands

the equatorial parts of its ecto-apertures; the equa- In the placental locule, the pollen tubes resynthesize torially situated endo-apertures are deeply drawn their callosic plugs and pollen tube wall. The rate inwards and sealed by the margins of the adjacent of growth decreases and consumption of probably exine. By contrast, a parasyncolporate pollen grain glycoproteinsoccurs. accommodates volume reduction by invaginating the A high number of pollen tubes can penetrate the polar parts; the endo-apertures remain superficial. micropyle of the ovule. By adding small crystals of Although experimental evidence is lacking, it is DAPI in the exudate droplet, the transport of the assumed that the different endo-aperturepositions in pollen tube nucleus and the sperm cell nuclei can be the dehydrated state have different protective values, followed within the ovule. It reveals that the two parasyncolporate pollen being more susceptible sperm cell nuclei are transferred to one of the syner- to desiccation. This might be the relevance of the gids between 8 and 15 h. The fusion of one of the hypothesized transformation of parasyncolporate to sperm nuclei with the egg cell nucleus takes place after colporateunder drier climatic conditions.

36 h. In the caseof multiplepollen tube penetrationof

the micropyle, the sets of nuclei of some pollen tubes

Ultrastructure and Morphometry of remain outside the micropyle, or in the micropylar

canal, or they can be observed between the nucellar Tobacco Pollen Tubes tissue and the first integument. G.P.J. Rongen. DepartmentofExperimental Ultrastructural studies show that 2-5 h after Botany, University of Nijmegen,Toemooiveld 1, pollinationthe micropylar canal is still empty and the NL-6525 ED Nijmegen, The Netherlands egg apparatus is still developing(Franssen-Verheyen

Growth of tip growing cells like pollen tubes is & Willemse, 1990, Acta Bol. Neerl. 39: 53-63). How-

8 supposed to concur with specific organelle distri- ever, h after pollination the pollen tubes have

the the butions, especially in growing tip (Steer & Steer entered raicropyle but are seldom in one of the 164 MEETINGS

synergids. At this time a viscous exudate is visible minate in hormone-free medium, but are blocked in

around the pollen tube. This dropletis formed during scutellum and shoot meristem formation. In 2,4D

the production of the stigmatic exudate and slowly medium they form embryogenic callus again. In fact,

increases in volume. embryogenic callus consists of globularstage somatic

After the pollen tube arrives in the micropyle, the embryos, linked by undifferentiated tissue. The latter

nucellar cells the of the bordering tip egg apparatus growinto maturesomatic embryos when transferred to

get small lipiddroplets. The nucellar cell wall adjacent maturation medium. The histology of these somatic

to the of the shows electron is similar to the tip egg apparatus an embryos remarkably histology ofzygo-

dense The filliform to become tic Characteristic layer. apparatus seems embryos. structures, such as coleop-

narrower. In the synergids the amount of starch tile, coleorrhiza, leaf primordia, peribleme,plerome,

decreases. root meristem, adventitious root meristem, scutellum,

These phenomena indicate an activation of the shoot meristem and scutellar node, areall present. The

ovule during the progamic phase. The observed embryois loosely attached with suspensor-like cells to phenomena point to either ovular reactions in selec- the callus aggregate. This aggregate seems to provide tion and of ovular acceptance one pollen tube, or to signalsthat stimulate the formation ofa normal shoot, incompatibility. includingscutellum and coleoptyle.

In the suspension, cell clusters developinto globular

Freeze Fracture Analysis of Embryogenic stage somatic embryos. The clusters were compared to the non-dividingelongated cells of the suspension by Suspension Cells and Histology of Somatic means of freeze fracturing. Both types of suspension Embryos of cells contain low number of terminal a very globules A.M.C. Emons and H. Kieft. Departmentof Plant and particle rosettes in their plasma membranes, as Cytology and Morphology, Wageningen 2 texture infrequentas 0-3-1 pm . The cell wall ofclus- Agricultural University, Arboretumlaan 4,6703 BD ters isdifferent from that ofelongated cells. Walls ofthe Wageningen, The Netherlands cluster cells have highly organized textures, consisting

In the presence ofthe synthetichormone 2,4D, young of lamellae with microfibrils in alternatingdirections. stages ofsomatic embryos ofmaize genotype 4C1 (de- Elongated cells have randomly oriented microfibrils in

velopedby Sandor Morocz and Denes Dudits,Szeged, the part of the wall deposited during elongation as

Hungary) are formed from calli in liquid medium. single cells. Plasmodesmata are abundant in cluster

Single somatic embryos, unattached tothe callus, ger- cells, but scarce in elongatedsingle cells.

MEETING OF THE SECTION FOR FERTILIZATION RESEARCH

IN PLANTS ON 5 OCTOBER 1990

Somatic and Sexual Interspecific Hybrids hybrids were obtained by electrofusion. The somatic

hybrids were tetraploid (8), hexaploid (3), octaploid between Solanumtuberosumssp. tuberosum (1) or mixoploid (1). Male and female fertility and (L.) and S. circaeifolium ssp. circaeifolium chromosomal configurations during male meiosis

(Bitter) were studied using some of the diploid, triploid and K.M. Louwes and W.M. Mattheij. Centre for Plant tetraploid hybrids. The percentage ofstainable pollen Breeding Research (CPO), P.O. Box 16, in the sexual hybrids varied from less than 5 to more Wageningen,The Netherlands than 50%. Not only normal haploid pollen but also

micropollen and unreduced pollen were observed.

Sexual hybrids between the Bolivian diploid wild The somatic hybrids (tetraploid) showed nostainable

Solarium In female for the species circaeifolium ssp. circaeifolium (crc) pollen. crossing experiments, fertility

and of the cultivated S found be rather for the diploids potato . tuberosum ssp. diploidswas to good, triploids tuberosum (tbr-2x) were obtained by crossing. Forty- it was of an acceptable level and for the tetraploids seven hybrids resulted from 850 pollinations, eight it was found to be very high. The cross (crctbr-

and diploids 39 triploids. Crc has two very typical 3x) x (tbr-2x), gave aneuploidprogeny with a mean of nucleolar and chromosomes as the triploid hybrids 30 chromosomes. Metaphase I analyses showed the contain two crc nucleolar chromosomes, they are occurrence of many univalents in the diploid, 2-3

from unreduced cell of trivalents thought to originate an egg crc per cell in the triploid and some trivalents

and a normal haploid male gamete from tbr. Somatic and/or quadrivalents in the tetraploidhybrids. These MEETINGS 165

Table 1. The various media used for the in-vitro techniques

-1 Culture Medium (%) NAA (mg I ) PH DAP

Ovary-slice* MS 9 1 60 7^10

Ovule MS 5 01 5-5 40-80

Embryo MS 2-4 0-1-0 001 50 40-80

Ovary** MS 7-8 5-8 2

*One sectioned in 6-10 slices. ovary transversely **In-vitro pollinationculturing whole ovaries.

results are in agreement with earlier reports, where no been isolated from a cDNA library made against structural genome differences were found between mRNA from mature pollen. Expression of KW303 diploid potato species and recombination is has been detected in pollen and not in other vegetative common. or generative tissues. mRNA hybridizing to this clone

ispresent in pollen from Digitalispurpurea and Lilium

longiflorum. The first transcription of the KW303 In-Vitro Pollinationof Lily takes after gene place microspore mitosis. Transcripts J.M. Van Tuyl, M.P. Van Dien, M.G.M. Van Creij accumulate thereafter. and R.J. Bino. Centre for Plant Breeding Research further For characterization, the base sequence of (CPO), P.O. Box 16, Wageningen, The Netherlands determined. No pKW303 was protein coding open

reading frame has been found, as 200 bp at the 5' side The use of a complete and integratedin-vitro pollina- still have be A database search has fertilization to sequenced. tion, and embryo rescue system was

revealed no examined in lily. By combining pollinationtechniques homologuesgenes yet. The localization of the K.W303 gene to overcome pre-fertilization barriers with in-vitro transcripts

was studied by means ofin situ hybridization. As the methods to overcomepost-fertilization barriers, both

resolution of a conventional light microscope was under fully controlled conditions, interspecific lily too low, a confocal laser scanning microscope crosses could be made more efficiently. In-vitro cut-

(CLSM) was used for studying the results. The style pollination and in-vitro grafted style techniques CLSM produces images ofthe reflection of the silver were developed and applied on various interspecific

grains in the light sensitive emulsion that are more crosses using Lilium longiflorum,and both Asiatic and pronounced than those from the transmission light Oriental hybrids as the parents. In addition, methods

It can visualize the silver grains within a for culture and ovule culture microscope. ovary culture, ovary-slice 1 0 pm thick layer of a 10 pm thick section and thus were generated. Ovule swelling score in ovary cul- visualize of the total sequentially parts coupe. Im- ture was used to evaluate media effects on ovule

produced on the CLSM can be stored on an development. ages optical disk for future analysis. Preliminary results The optimal media and moment of application using the CLSM localized KW303 RNA within the (in DAP =days after pollination) developed for the vegetative cell. Other localization experiments are in in-vitro methods are summarized in Table 1; MS is the

progress. standard Murashige & Skoog medium. Northern blot analyses indicated transcription of

KW303 within the first two hours of germination.In of Localization, and Analysis Expression order to prove de novo synthesis of KW303 RNA a

Nucleotide Sequence ofthe Pollen Specific modified ‘run on’ transcription assay was developed.

The pollen grains were allowed to germinate cDNA Clone pKW303 in 3 the of H-Uridine. The newly synthesized K. Weterings. Departmentof Molecular Plant presence

(labelled) KW303 RNA was isolated by means of Physiology, Catholic University of Nijmegen, to liquid hybridization an antisense 303 probe, and Toernooiveld, 6525 ED Nymegen, The Netherlands this was followed by precipitation. For detection a

the of the male of the isolated Through development gametophyte fluorograph K.W303 RNA was made appears to be simple, there are 20,000 genes being after electrophoresis on a denaturingacrylaraid/urea

The molecular that form the slab this method of the expressed. processes gel. Using transcription

For KW303 could be basis of pollen development were studied. this gene detected within the first hour of

cDNA clone has purpose a pollen specific (pKW303) germination. 166 MEETINGS

the The Interactionof Pollen Tube Growth and In stylar fluid the protein pattern of the stylar

proteins showed no differences before and after Protein Patterns during the Progamic pollination.In the pattern, four proteins derived from Phase in Gasteria verrucosa (Mill.) the pollen tubes could be detected 3 h after pollina- H. Duval tion. In the placental fluid, three proteins derived from M.T.M. Willemse. of Plant Department Cytology the pollen tube could be detected 7 h after germination and Morphology,WageningenAgricultural at the same location on the gel as in the styler University, Arboretumlaan 6703 BD Wageningen, 4, fluid. Two spots present in the unpollinatedsample The Netherlands disappeared, however; oneprotein at lower pH and a

The exudate droplet on the stigma of Gasteria group ofwhat are probably glycoproteins.

is the for After Pollen in the of about 8% of verrucosa signal receptivity. pollina- germination presence tion, the amount of placental fluid increases and the placental fluid showed a breakdown ofsome proteins fluid in the hollow forms continuous of pathway style a including the group glycoproteins. In this pattern connection fluid. with the placental Samples of stylar the group of proteins which were present only in the

medium and medium The function ofthis last of fluid,placental fluid, pollen germination reappeared. group homogenizedgerminatedpollen were centrifuged and proteins is till now unknown. their supernatants were concentrated with an ultra- Duringpollen tube growth the stylar fluid proteins

in free 10,000 NMWL filter (Millipore). Concentrated are probably not used but the placental fluid there is samples of the native proteins were separated in a breakdown of what are probably glycoproteins. The the first dimension 4 6-5 and in the second excretes to these of by IEF, pH pollen enzymes digest type pro-

dimension on a 8-25% gradient PAGE gel on a teins. Addition of about 8% placental fluid to the

Pharmacia system. pollen germinationmedium induces an increase in the

The protein pattern of the in-vitro germinated rate ofpollen tube growth.

No pollen and its exudate in the medium showed one relationship between this and the ovular

of in the medium. in Gasteria could be made. group proteins present only pollen incompatibility not yet

MEETING OF THE SECTION FOR PHYTOPATHOLOGY ON 17

JANUARY 1991

Immunolocalizationof β5-1,3-glucanase and with a chitinase and a (i-1,3-glucanasc isolated and

purified from leaves which were inoculated Chitinase in Compatible and Incompatible with Cladosporium fulvum. We found that chitinases Interactions between Tomato and the and P-1,3-glucanases were localized in the cytoplasm Cladosporium fulvum of infected plants in both compatible and incompat- J.P. M.H.A.J.Joosten and P.J.G.M. De Wubben, ible interactions. However, only in the incompatible Wit. Department ofPhytopathology, Wageningen interaction a strong accumulation of the proteins in Agricultural P.O. Box 8025, 6700 EE University, electron dense particles in the vacuoles was found. Wageningen,The Netherlands Furthermore, we found that chitinases and P-1,3-

glucanases were localized around the stomata when In much attention has been the recent years paid to the plants were grown under suboptimalconditions. role of plant hydrolases which are induced during these From preliminary results we cannot yet con- the defence response against fungal pathogens. The clude that chitinases and P-1,3-glucanases play a p-1,3-glucanases and chitinases have been studied primary role in the defense of tomato against these able extensively as enzymes were especially to Cladosporiumfulvum. degradeisolated fungal cell walls, and torelease glyco-

act sidic fragments which consequently could as Xylem and PhloemTransport of elicitors of other defense responses. Tomato plants, Fluorescent Dyes to and into Nematode- infected by the fungus Cladosporiumfulvum. also Induced Giant Cells in Tomato respond with the production ofp-l,3-glucanases and R. Dorhout*{, F.J. Gommers* and C. Koll6flel{. chitinases. In incompatible interactions the chitinases •DepartmentofNematology, Wageningen and 3-1,3-glucanases accumulate more rapidly than in AgriculturalUniversity, Binnenhaven 10,6709 PD compatible ones. These findings suggested that these and Research the active Wageningen, {Transport Physiology enzymes mightplay a role in defense. Group, University of Utrecht, Botanical Laboratory, To get a better understanding of the actual role of Nieuwstraat 3512 PN The these in this Lange 106, Utrecht, hydrolytic enzymes interaction,we inves- Netherlands in situ tigated the localization of these proteins in

infected tomato leaves. For this purpose we used The fluorescent dyes, fluorescein (F, pAT=4-9) and

antisera obtained from rabbits which were injected carboxyfluorescein (CF, pA’ = 4 0), were used to study MEETINGS 167

xylem and phloem transport to and into giant cells, strain YSMV induces necrotic local lesions and induced by Meloidogyne incognita. The uptake of systemic yellowing of the leaves. By making pseudo- these dyes by plant cells can be accounted for by an recombinants between the afore mentioned strains, acid-trap mechanism, which assumes that their entry symptom formation in tobacco has been assigned to mainly occurs by passive diffusion of the undis- RNA3 (Dingjan-Versteegh et at. 1972, Virology 49, sociated form of the acids. Passage of the anions 716-722). Nucleotide sequences of RNA3 of both

the construction of across cell membranes occurs very slowly. strains are known, and cDNA

Xylem transport was studied by applying the dyes clones of RNA3 from which infectious transcripts can to infected tomato root systems of which some root be synthesized, allowed the investigation of parts of tips were removed and replaced by polyethylene cups the molecule involved in this differential symptom

DNA containing one of the dyes. Phloem transport was formation. fragments were exchanged between investigated by applying the dyes to the adaxial sur- the cDNA clones ofthe strains and transcripts thereof

of tomato to the this face of nearly full grown leaves infected inoculated transgenic plants. In way, a plants, as described previously (Grignon el al. 1989, single amino acid substitution (Gln(29) to Arg) in

Am. J. Bot. 76: 871-877). Afterone day ofincubation, the iV-tcrminal part ofthe coat protein was found to handcut sections of the root systems were made and induce the local lesion phenotype of strain YSMV.

For viewed with an epifluorescence microscope equipped Systemic symptom formation was more complex. with suitable filter combination sets. most hybrids inducing local lesions, a severe delay

When the in the in observed and dyes were applied a cup at apical systemic yellowing was yellow cut end ofa root, they were transported via the xylem patterns in some casesdiffered from wild type YSMV

sites vessels in shoot direction. At infection F ac- symptoms(giving yellowingalong the nerfs, instead of cumulated in the giant cell complexes, whereas CF between them). accumulation only occurred when the phloem also fluoresced. Leaf surface application of the dyes resulted in phloem CF was transported to transport. Bacteria Associated with Sheath and Grain F the roots within one day, whereas had a high lateral Discolourationof Rice Once CF the escape. was transported through M.F. Van Outryve, B. Cottyn, M. De Cleene, T.W. infected root, the dye was only visible in the phloem Mew and J. Swings. Laboratory for Microbiology and in the giantcells. Since CF did not accumulate in and Microbial Genetics, State University Ghent, the giant cells when applied via the apoplast, it was Belgium, and Plant Pathology Division, concluded that there is a symplastic pathway between International Rice Research Institute, Los Banos the phloem and the giant cells. So, the giant cells Philippines seem to have the capacity to accumulate nutrients

from the xylem as well as the phloem via nutrient- Several bacterial and fungal pathogens have been proton-cotransport and via plasmodesmatal trans- found in association with sheath and grain discolour- port respectively. ation ofrice. Among the bacteria, species ofthe genera

Pseudomonas and Erwinia are known to cause such

symptoms. Inthis project we want to identify the bac- Mosaic Virus Replication in teria associated with symptoms of sheath and grain Transgenic Plants Expressing Viral discolouration of rice and design fast and reliable

Replicase Genes: Role of the Coat Protein identification methods for them.

Gene in the Virus/Plant Interaction During the wet season of 1988 and 1989, plant

material of sheath and dis- A.C. van derKuyl. Department of Biochemistry, showing symptoms grain

Gorlaeus Laboratories,Leiden University, P.O. Box coloration were sampled throughoutthe Philippines.

thousand bacteria 9502,2300 RA Leiden, The Netherlands From these plants, over seven were

isolated and checked for pathogenicity on rice seed-

Transformation of Nicotiana tabacum cv, Samsun lings and plants in booting stage. Two hundred and

1 NN with RNAs and 2 (encoding subunits of the thirty-six isolates were found to be pathogenic.

viral replicase) of the tripartite genome of alfalfa At present the activities at the laboratory of

mosaic virus (+ rise and microbial of the State (AIMV, a )-RNA virus) gave microbiology genetics

the third ofGhent focused the identification to plants efficiently replicating genome University are on

segment, RNA3, encoding a protein involved in cell- of the isolates and the design of fast identification

to-cell transport of the virus and the viral coat systems which in future might be used by plant

services. first chromato- protein. quarantine In a stage gas

Symptom formation by AIMV in N. tabacum is graphic analysis of the fatty acid methyl esters and

dependentuponthe particular strain used. The Leiden standardized micromethods for phenotypic tests are

isolate of strain 425 induces mild while examined for this a chlorosis, purpose. 168 MEETINGS

A Preliminary Study to Identify Soil Biotic Identificationof ArmillariaSpecies using

Factors Involved in the Degeneration of Mating Experiments

Ammophila arenaria (marram grass) B.C. van Dam. ‘De Dorschkamp’, Institute for

for Forestry and Urban Ecology, P.O. Box 23, 6700 AA P.C.E.M. van der Goes, Institute Ecological Wageningen,The Netherlands Research, P.O. Box 40, 6666 ZG Heteren, The

Netherlands In Europe five species of Armillaria occur. A. mellea

A. A. and obscura may attack healthy trees whereas arenaria Ammophila (marram grass) is a species that bulbosa only attacks weakened trees. A. borealis fixes and stabilizes sand and occurs mainly in the A. and cepistipes are saprophytes. Variation in the Dutch coastal foredunes. A. arenaria is most vigorous morphology of fruiting bodies is considerable and on the seaward slopes, where it is buried regularly by identification using basidiocarps is not always fresh windblown sand. However, it degenerates when reliable. sand accumulation diminishes. be Degeneration may Interfertility experiments have proved to be a

due to a harmful biotic factor in the soil. Pot exper- useful tool in the of Armillaria. Haploid iments showed that the growth of A. arenaria in soil cultures obtained from basidiospores produce white from its root zonewas significantlyimproved when a to light brown fluffy, aerial mycelia, whereas fully nematicide was used. The same study also suggested compatiblepairings produce dark brown flat crustose that fungi may be involved. It was suggested that the mycelia. colonization ofwindblown sand enables A. arenaria to Haploid tester strains ofthe five species weremated maintain because ofthe escape from harmful vigour 44 the with isolates obtained from cap (diploid) of soil organisms. basidiocarps and single isolates (haploid) from of this is The purpose study to identify potentially the same basidiocarps growing in oak stands. Thirty- harmful nematodes and fungi in the root zone of A. five isolates appeared to be A. obscura, six isolates arenaria. A survey was conducted at five locations were A. bulbosa and two isolates were A. mellea. One along the Dutch coastal foredunes: the isle of isolate could not be identified. A. borealis and A. Voorne, Schouwen and Goeree (all lime-rich dunes), cepestips were not found. Callantsoog and the isle of Texel (both lime-poor Diploid x haploid matings (i.e. Bullet Phenomenon) dunes). At all locations samples were collected from were compared with haploid x haploid matings. the root zone ofvigorous and ofearly-decliningstands to Diploidx haploid mating reactions were difficult of A. arenaria. Nematodes and fungi were isolated score because one mate already possesses crustose from soil and roots, and identified. mycelium. Generally however, they lead to the same The plant parasitic nematodes found were identification. It was very important that the tester Heterodera sp. (avenaegroup),Meloidogynemaritima. strains isolated in the old were same year; one year and Telotylenchus sp., Tylenchus spp., Pratylenchus results. It concluded that testers gave confusing was Nematode numbers low those sp. were ascompared to A. obscura was prevailing in oak stands. in agricultural systems and there was much variation

The results the among sample replicates. support hypothesisthat nematodes may not be the only group Mechanisms Involved in Control of

ofsoil organisms involved in the degeneration of A. Fusarium Wiltof Carnation by Fluorescent arenaria. Pseudomonas spp. Sixty-three fungal species were found. They could B.J. Duijff, J.W. Meijer, P.A.H.M. Bakkerand B. be classified into four of groups: one group fungi Schippers. Dept, of Plant Ecology and Evolutionary seemed to be specific for lime-rich foredunes, Biology, Section ofPlant Pathology,University of another for lime-poor foredunes. A third group Utrecht, Willie Commelin Scholten

group was regularly found in both lime-rich and PhytopathologicalLaboratory, Baarn, The lime-poorforedunes and a fourth group was only in- Netherlands

cidentally isolated. The fungi with a possible role in

be found in the third To the of the control of soilborne degenerationmay group men- improve efficacy tioned, This preliminary study suggests that the plant pathogens by treatments with fluorescent fungi involved in the degeneration of A. arenaria pseudomonads, the mechanisms involved have to

be: Fusarium culmorum, be elucidated. Wilt disease of caused may Apiospora montagnei. carnation, by

Harzia Microdochium Phoma Fusarium f. dianthi used as acremonioides, bolleyi, oxysporum sp. (Fod), was

of spp., Trichothecium roseum and several Ulocladium a model to study the involvement siderophore

mediated for antibiosis induced spp. competition iron, and

The possible role of nematodes, fungi, and their resistance in disease suppression by two strains interactions in the degeneration of A. arenaria will be of Pseudomonas: P. sp. WCS4I7r and P. putida studied further by inoculation experiments. WCS3SBr. MEETINGS 169

The purified siderophore of both strains inhibited gate whether polygalacturonases (PC’s) could be germination of conidia of Fod. The iron saturated such a parameter we stem-inoculated 11 carnation siderophore inhibited germinationto a much lower cultivars which differed in resistance level. This

7 degree. These results suggest that siderophore inoculation was performed with a suspension (10 mediated for iron be involved in the conidia ml of Fusarium f. dianthi competition can ') oxysporum sp. suppression of Fusarium wilt. Involvement of side- race 2 on rooted cuttings which were planted in soil rophore mediated competition for iron was also 4 weeks before. During the experiment, disease observed The inhibition ofFod both indexed 12 weeks after on agar plates. by symptoms were weekly up to

Pseudomonas strains was reduced with increasing iron inoculation. Four weeks after inoculation, PC’s content of the medium, and a non-siderophorepro- were extracted from four stem pieces which were ducing (sid) Tn5 mutant of WCS358 did not inhibit 5 cm in length (taken directly above the inoculation

Fod. The sid Tn5 mutants of WCS417r, however, site) 4 weeks after inoculation. Electrophoresis of still inhibited Fod, suggesting the production of an these extracts showed four PG isoenzymes on gel antibiotic by this strain. which were not present in extracts from water inocu-

Treatment of carnations with WCS417r resulted in lated plants. These PC’s are most likely of fungal - difference a significant disease reduction. The sid mutants of origin. Most striking was the observed this strain reduced disease incidence less than their between the 11 cultivars. These cultivars appeared to wild type. WCS358r reduced disease incidence not have less PG activity as the resistance level

- To significantly. Its sid mutant did not reduce disease increased. quantify these differences we used a

incidence. and cupplate assay a spectrophotometrical assay.

The ability of the Pseudomonas strains to induce Both methods clearly indicated a significant positive

resistance against Fod in carnation, was studied by correlation between PG activity and disease index

spatially separating the bacteria (on the roots) and and a significant negative correlation between PG

reduced within the pathogen (in the stem), WCS417r disease activity and percentage resistant plants a

incidence significantly; WCS358r did not. clone. From these results was concluded that

From these results it is concluded that in addition measurement of PG activity within stems of infected

induced mediated carnations could be useful tool determine the to resistance, siderophore compe- a to

tition for iron and antibiosis could also be involved resistance level of a cultivar.

in suppression of Fusarium wilt of carnation by

Pseudomonas WCS4l7r. The of sp. suppression

Fusarium wilt by Pseudomonas putida WCS358r seems Does a Vascular Fungus Elicit an Induced

to depend on siderophore mediated competitionfor Response in Tomato Plants, thereby also

iron only. The multiple mechanisms of WCS417r may Affecting Fecundity ofthe Two-Spotted explain its more efficient control of Fod, compared Spider Mite? with WCS358r. P.H.J. Jongebloed, D M. Elgersma* and M.W.

Sabelis. Department ofPure and Applied Ecology,

Section Population Biology, University of Relation Between In Vivo Productionof Amsterdam, Kruislaan 302, 1098 SM Amsterdam, Fungal Polygalacturonases and Resistance and “Willie Commelin Scholten Phytopathological Level ofCarnation Cultivars to Fusarium Laboratory, Javalaan 20, 3742 CP Baam, The

wilt Netherlands

E.A.M. Schoffelmeer, S. Toet and D.M. Elgersma.

Department of Molecular Cell Biology, Section Previous inoculation oftomato plants with a vascular

Plant of Fusarium f, Pathology, University Amsterdam, fungus ( oxysporum sp. lycopersici. race

Willie Commelin Scholten Phytopathological 1), caused a decrease in the oviposition rate of two-

Laboratory, Javalaan 20, 3742 CP Baarn, The spotted spider mites (Tetranychus urticae) on a

Netherlands Fusarium- susceptible: cultivar, but not on a Fusarium-

resistant cultivar. We conclude that the effect on mite

Breeding Fusarium-wilt resistant carnations is fecundity is due to the fungus affecting the plant’s food

the best the the effects this have the probably approach to overcome prob- quality, including may on

lem that is caused Fusarium f. of defensive rather than the by oxysporum sp, composition compounds,

dianthiin carnation culture. This resistance breeding plant’s active defense system. Since Fusarium seals

program is hampered by the lack ofa parameter, by off the xylem vessels, thereby causing wilting of the

which resistance can be measured in a fast and reli- susceptible tomato plants, the reduction in mite

able fashion, other than determining the percentage fecundity may well be due to drought stress in the

in clone inoculation which is leaves. In surviving plants a after some preliminaryexperiments we found an

To of in the environment strongly influenced by the environment. investi- effect severe drought stress root 170 MEETINGS

the of T. that started the activation of on oviposition rate urticae. suggesting to study gene by means

be two-dimensional our hypothesis may right. polyacrylamide gel electrophoresis

of in-vitro translation products in two near isogenic

lettuce lines (F8 backcross). One line is susceptible to Growth and Oospore Formationoftwo

the physiological race NL12 and resistant to NL15, in Pythium spp. Causing Cavity Spot the other line is susceptible to NLI5 and resistant to NL12. At least five differences in translation products I. Blok. Research Institute for Plant Protection, were found between the mRNAs isolated from the two

P.O. Box 9060,6700 GW Wageningen,The near isogenic lines. No differences were found in

Netherlands translation products of mRNA isolated from water,

NL12 and NLI5 inoculated leaves within one day Cavity spot is a disease which can be caused by after inoculation. Therefore we conclude that dif- two species of Pythium: P. violae and P. sulcatum. ferences in activation be restricted the gene may to Both pathogensare seldom found in carrots from the lettuce epidermal cell layer. mRNA will further be same field. P. sulcatum is more often isolated from isolated from epidermal cell layers stripped off leaves muck while violae carrots grown on sandyor soils, P. at different time periods after the inoculation with the prevails in clay soils. fungus. The pH influences the growth of both fungi on agar media in the Growth ofboth does same way. species not is limited at 5 0 the occur or very pH dependingon Gene Expression in Phytophthora infestans medium used. The optimum temperature, however, During Pathogenesis on Potato differs for both fungi. P. violae shows most mycelial C.M .J. Pieterse. DepartmentofPhytopathology, whereas sulcatum growth on agar at I9-20°C, P. WageningenAgricultural University, P.O. Box 8025, grows best at 23-24°C. Oospore formation is also 6700 EE Wageningen,The Netherlands quickest at these respective temperatures. Whether

P. violae media produces oospores on agar depends Potato late blight caused by the fungus Phytophthora on the composition of the media, wateragar being infestans (Mont.) de Bary (Oomycetes), is one ofthe the most satisfactory. P. sulcatum produces easily most important diseases ofpotato. Leaves and tubers

oospores. are readily infected by P. infestans and the coloniza-

To free oospores from mycelium, sonication as well tion process is very rapid and extremely destructive as through snails promising. passage appears for the tissue. The molecular basis underlying patho-

genicity of P. infestans is poorly understood. In order

to more insight in the we In-vitro Translation Products from gain processes involved, are the of P. studying gene expression infestans during Compatible and Incompatible pathogenesis onpotato. Combinationsof Bremia lactucae and It can be assumed that the establishment of a

Lettuce pathogenic relation between the potato plant and P.

H. vanPelt-Heerschap and O. Smit-Bakker. infestans involves the mutual interference in cellular

Research Institute for Plant Protection, processes of both partners. Defence responses of

P.O. Box 9060, 6700 GW Wageningen,The colonized host tissue are relatively well studied, but

Netherlands nothing is known about changes in the metabolism of

the pathogen duringgrowth in the plant. It is tempting

Bremia P. lactucae Regel, the causal organism of lettuce to assume that in the potato-; infestans interaction,

downy mildew, is a frequently occurring pathogen of host factors induce physiological responses in the

The disease is for field and glasshouse lettuce crops. pathogen which are necessary pathogenesis and

In disseminated by means of conidiosporangia. the which are mediated by pathogenicity genes. The

of in infection process, no distinction in the behaviour identification and characterization of planta

induced of therefore will lead either fungus or plant for compatible or incompatible genes P. infestans to a combinations have been found prior to penetrationof better understanding of the molecular basis under- the in the lettuce cell The of the To this end epidermal layer (In: lying pathogenicity fungus. a

D.M. Downy Mildews, ed. Spencer, 1981). The contact genomic library ofP. infestans DNA was differentially

ofthe fungus with the host plasmalemma,or later the screened using cDNA from mRNA of the fungus

in vitro formation of the fungal secondary vesicles probably grown and cDNA from mRNA of infected contributes to differences in compatible and incom- leaves as probes. Several differentially hybridizing patible combinations. Studies of the differences in clones containing putative in planla induced P.

and the from gene activation in both the fungus plant, for infestans genes were isolated the P. infestans

The DNA of of the both resistant and susceptible interactions, may show genomiclibrary. sequence some in

been gene products which determine the specificity. We planta induced genes has determined. Two of MEETINGS 171

these encode and calmodulin in several cellular The appear to übiquitin important processes. expression

Both conserved in of the and is respectively. genes are highly genes in planta in vitro currently being eukaryotic organisms and are reported to be involved studied.