Micromorphology of the petals of the invasive weed,Oxalis pes‐caprae

The alien, seedless Oxalis pes‐caprae has spread and colonized many areas of the Mediterranean Basin, relying on vegetative reproduction. The flowering of O. pes‐caprae is greatly accelerated by its exposure to sunlight. When the sun is shining, both sides of the petals of the funnel‐shaped, open flowers of O. pes‐caprae are exposed to the ambient conditions. In cloudy weather, only some portions of the abaxial petal surfaces of the trumpet‐shaped, closed flowers of O. pes‐caprae are exposed to the ambient conditions. The micromorphology of the petals of O. pes‐caprae was imaged by using light, scanning and atomic force microscopy. In O. pes‐caprae, conical cells are found only on the adaxial epidermis of the petals, which also consist of a narrow mesophyll with a loosely arranged parenchyma and convex cells on their abaxial epidermis. High‐resolution imaging of the petal surfaces, using atomic force microscopy, revealed that the epidermal cells are further ornamented by submicron sculptures, indicating a different roughness, density, and arrangement of the folds between the adaxial and abaxial sides of the petals. Submicron sculpturing increases the surface area of the adaxial epidermal cells of the petals and the distances between the folds are almost equal to the visible waveband. On the abaxial epidermal cells, the distances between the folds are smaller than the subwavelength spectrum. The high and the negligible values of roughness that were obtained on the adaxial and the abaxial surfaces might facilitate the capture and the reflection of light, respectively.     

花卉杂色花的研究

 在云南昆明、玉溪、楚雄、曲靖等地区,我们发现四种花卉有杂色花。所谓杂色花是由病毒侵染而引起的一种特殊花,它鲜艳美丽比无病的花还好看。这种花早在十六世纪于荷兰种植的郁金香上发现,即在花瓣上产生一种白色花斑或条纹,比一般的单色花更加美丽,于是身价倍增,当时人们互相争购,因此把此时期称为"郁金香疯狂时代(Tulipomania)",后来经病毒接种试验证明是由郁金香碎色病毒(TBV)引起的。     

Histopathology of the initial stages of the interaction between rose flowers and Botrytis cinerea

The early stages of the interaction between flowers of the cut rose cv. Sonia andBotrytis cinerea was investigated by scanning electron microscopy and light microscopy. Infection of petals by conidial germ tubes evoked a susceptible reaction. In contrast to general findings nutrient addition to the inoculum was not a prerequisite for this phenomenon. At the lower side of germ tube tips the cuticle was penetrated by infection pegs. Already at this early stage of the infection process, the infection sites were macroscopically visible as scattered white spots. After penetration, pegs enlarged to form infection hyphae, which invaded the periclinal wall of outer epidermal cells. At those sites, the petals formed outgrowths of variable appearance at their abaxial side. Thee outgrowths consisted of remanants of collapsed epidermal cells and of infection hyphae. Subsequent intra- and intercellular growth of hyphae led to a collapse of epidermal and mesophyll cells. The symptoms described generally developed within 24 h. After subsequent incubation the lesions became necrotic. Eventually, the necrosis would spread leading to the death of whole petals.     

Light and Scanning Electron Microscopy Studies on the Infection of Oriental Lily Leaves By Botrytis Elliptica

Light, scanning electron and fluorescent microscopy were used to observe the infection process of Botrytis elliptica on leaves of oriental lily (cv. Star Gazer). At 20 °C and 100% relative humidity, conidia germinated on both adaxial and abaxial foliar surfaces, but germ tubes failed to invade epidermal cells on the adaxial surface. On abaxial surfaces, short (< 20 m) swollen germ tube appressoria penetrated through stomatal openings (19%), through the epidermis near guard cells (52%), or directly through epidermal cells (29%). Esterase activity was detected on germ tubes and conidia after 6 h of incubation, and deformation of the cuticle on abaxial surfaces of lily was observed surrounding infection sites. By 3 h after inoculation, almost 70% of the conidia had germinated, but no penetration was observed. At 6 h after inoculation, almost one-third of germinated conidia had penetrated epidermal cells, and water-soaked lesions were associated with 20% of the penetrations. By 9 h after inoculation, approximately 60% of the germinated conidia had penetrated plant tissues, and water-soaked lesions were associated with 60% of the infections. Fluorescent microscopy with a specific fungal stain allowed assessment of successful infection and visualization of sub-epidermal hyphae. We conclude that penetration of abaxial foliar surfaces of oriental lilies by B. elliptica occurs via short swollen germ tube appressoria mostly near stomata.     

Early stages of infection of rapeseed petals and leaves by Sclerotinia sclerotiorum revealed by scanning electron microscopy

The primary ascospore inoculum of Sclerotinia sclerotiorum initially infects rapeseed (Brassica napus var oleifera) via petals. Infected petals fall onto leaf surfaces, resulting in infection of those organs. A scanning electron microscopy (SEM) study of this process was undertaken to elucidate the host-parasite relationship and to determine the best plant organ for detection by serology of early field infection as an aid to disease forecasting and cost-effective disease control. The behaviour of ascospores deposited on young petals and on leaves was compared. Ascospores were deposited by inverting a mature apothecium above either a leaf disc, a young petal or young petal placed on a leaf surface. Spore germination, host penetration and colonization were examined by SEM. On young petals, the following steps in pathogenesis were observed: ascospore adhesion and germination, penetration of the host from short germ tubes and collapse of epidermal cells. Petals were then covered with extensive mycelium. From these sites, the mycelium invaded leaf tissues and infection proceeded. In contrast, ascospores landing directly on leaf surfaces failed to germinate. The role of petals as sites of pre-election in the aetiology of the disease is discussed in relation to the published literature.     

Induction, Regulation, and Role in Pathogenesis of Appressoria in Monilinia fructicola

ABSTRACT Monilinia fructicola, which causes brown rot in stone fruit, forms appressoria on plant and artificial surfaces. On nectarine, the frequency of appressoria produced by conidial germlings depends to a large degree on the stage of fruit development, with numerous appressoria formed on immature (stage II) nectarine fruit, and no appressoria observed on fully mature fruit (late stage III). On polystyrene surfaces, appressorium formation was increased from <10% of germinated conidia to >95% of germinated conidia when the conidia were washed to remove residual nutrients and self-inhibitors. M. fructicola appressorium formation also appears to be regulated by the topography of the plant surface. On fruit, appressoria formed on stomatal guard cell lips, on the grooves of lateral cells adjacent to stomata or between two epidermal cells, and on the convex surfaces of epidermal cells. Pharmacological effectors indicate that cyclic AMP-, MAP kinase-, and calcium/calmodulin-dependent signaling pathways are involved in the induction and development of appressoria. KN-93, an inhibitor of calmodulin-dependent protein kinase II, did not inhibit conidial germination but did inhibit appressorium formation and brown rot development on flower petals, suggesting that appressoria are required for full symptom development on Prunus spp. petals.     

Relations between glasshouse climate and dry weight of petals,epicuticular wax,cuticle, pre-harvest flowering period and susceptibility toBotrytis cinerea of gerbera and rose flowers

Studies were conducted on the effects of seasonal levels of relative humidity, temperature, and total radiation, on dry weight of petals, on fresh weight of epicuticular wax and of cuticle of petals, on numbers ofBotrytis cinerea lesions in petals, and on preharvest flowering periods in gerbera and rose. No temporal relationships or significant correlations were found among dry weight of petals, weight of wax and cuticle of petals, and numbers of lesions on the petals. Temperature, relative humidity and total radiation did not correlate significantly with dry weight of petals, or with fresh weights of wax and cuticle of petals, except for a positive correlation between relative humidity and cuticle weight in the gerbera cultivar Delphi. No relation was found between weight of epicuticular wax and cuticle of petals and susceptibility of gerbera and rose petals toB. cinerea. The thickness of wax and cuticle on flowers did not seem to be an important factor influencing the susceptibility of flowers toB. cinerea. The seasonal pattern in number of lesions produced on the flowers byB. cinerea was related to the effects of relative humidity and radiation on infectivity of conidia of the pathogen on the flower surface but not to the effects on the susceptibility of flowers.     

Subcuticular-Intracellular Hemibiotrophic and Intercellular Necrotrophic Development of Colletotrichum acutatum on Almond

ABSTRACT The early infection and colonization processes of Colletotrichum acutatum on leaves and petals of two almond cultivars with different susceptibility to anthracnose (i.e., cvs. Carmel and Nonpareil) were examined using digital image analysis of light micrographs and histological techniques. Inoculated tissue surfaces were evaluated at selected times after inoculation and incubation at 20 degrees C. Depth maps and line profiles of the digital image analysis allowed rapid depth quantification of fungal colonization in numerous tissue samples. The results showed that the early development of C. acutatum on petals was different from that on leaf tissue. On petals, conidia germinated more rapidly, germ tubes were longer, and fewer appressoria developed than on leaves. On both tissues, penetration by the pathogen occurred from appressoria and host colonization was first subcuticular and then intracellular. On petals, colonizing hyphae were first observed 24 h after inoculation and incubation at 20 degrees C, whereas on leaves they were seen 48 to 72 h after inoculation. Intercellular hyphae were formed before host cells became necrotic and macroscopic lesions developed on petals >/=48 h and on leaves >/=96 h after inoculation. Histological studies complemented data obtained by digital image analysis and showed that the fungus produced infection vesicles and broad hyphae below the cuticle and in epidermal cells. In both tissues, during the first 24 to 48 h after penetration fungal colonization was biotrophic based on the presence of healthy host cells adjacent to fungal hyphae. Later, during intercellular growth, the host-pathogen interaction became necrotrophic with collapsed host cells. Quantitative differences in appressorium formation and host colonization were found between the two almond cultivars studied. Thus, on the less susceptible cv. Nonpareil fewer appressoria developed and host colonization was reduced compared with that on cv. Carmel.     

利用OWRK法预测桃叶表面润湿性能的研究

对桃叶表面润湿性能的预测方法进行了研究。应用OWRK法对不同生长期桃叶的近轴面、远轴面的表面自由能及其分量进行了测定。无论近轴叶面还是远轴叶面,随着桃叶生长期的增长,均表现出表面自由能减小,润湿性能变差,极性分量所占比例下降,色散分量所占比例上升等特性。这些变化与前人对于桃叶表皮蜡的蜡质含量、组份含量变化的研究成果相一致。说明用OWRK法测定桃叶表面自由能及其分量所得结果可靠,可以较好地描述桃叶表面的润湿性能和能量特征。     

PHYTOTOXICITY OF PARAQUAT ON WHITE AND GREEN HIBISCUS,SORGHUM AND ALPINIA LEAVES*

Summary. Paraquat was applied to while, variegated and green leaves of Hibiscus rosa sinensis L. Visual observations and moisture loss determinations revealed that the rate of tissue kill by paraquat was reduced in while tissue. Evidence from paraquat absorption and concentration studies indicated that phytotoxic differences were not due to amount or rate of absorption or protoplasmic susceptibility, but to differences in chlorophyll Content of treated leaves. Desiccants such as dinoseb, not dependent upon chlorophyll for rapid action, were equally affective on white and green leaves. The same reduced activity of paraquat on white leaf tissue was demonstrated for other species. Phytotoxicité du paraquat appliqué sur les feuilles blanches ou vertes de l'hibiscus, du sorgho et de l'alpinia     

Infection of wheat spikes by <Emphasis Type="Italic">Fusarium avenaceum</Emphasis> and alterations of cell wall components in the infected tissue

The infection process of Fusarium avenaceum on wheat spikes and the alteration of cell wall components in the infected host tissue were examined by means of electron microscopy and cytochemical labelling techniques following spray inoculation at growth stage (GS) 65 (mid-flowering). Macroconidia of the pathogen germinated with one to several germ-tubes 6–12 h after inoculation (hai) on host surfaces. The germ-tubes did not penetrate host tissues immediately, but extended and branched on the host surfaces. Hyphal growth on abaxial surfaces of the glume, lemma and palea was scanty 3–4 days after inoculation (dai) and no direct penetration of the outer surfaces of the spikelet was observed. Dense mycelial networks formed on the inner surfaces of the glume, lemma, palea and ovary 36–48 hai. Penetration of the host tissue occurred 36 hai by infection hyphae only on the adaxial surfaces of the glume, lemma, palea and upper part of ovary. The fungus penetrated the cuticle and hyphae extended subcuticularly or between the epidermal wall layers. The subcuticular growth phase was followed by penetration of the epidermal wall, and hyphae spread rapidly inter- and intracellularly in the glume, lemma, palea and ovary. During this necrotrophic colonization phase of the wheat spike, a series of alterations occurred in the host tissues, such as degeneration of cytoplasm and cell organelles, collapse of host cells and disintegration of host cell walls. Immunogold labelling techniques showed that cell walls of spike tissues contained reduced amounts of cellulose, xylan and pectin near intercellular hyphae or infection pegs compared to walls of healthy host tissues. These studies suggest that cell wall degrading enzymes produced by F. avenaceum facilitated rapid colonization of wheat spikes. The different penetration properties of abaxial and adaxial surfaces of the spikelet tissues as well as the two distinct colonization strategies of host tissues by F. avenaceum are discussed. The penetration and colonization behaviour of F. avenaceum in wheat spikelets resembled that of F. culmorum and F. graminearum, although mycotoxins produced by F. avenaceum differed from those of the latter two Fusarium species.     

Histopathology of postbloom fruit drop caused by Colletotrichum acutatum in citrus flowers

Postbloom fruit drop (PFD) is caused by both Colletotrichum acutatum and C. gloeosporioides and is a potentially serious disease in citrus that occurs when flowering coincides with rainfall. The fungus incites necrotic lesions in petals and stigmas leading to premature fruit drop and reduced yield. The mechanisms of infection and survival of the causal agents remain to be fully elucidated. In the present study, we investigated the histopathology of PFD caused by C. acutatum in the petals and stigmas of sweet oranges using electron and light microscopy. In the petals, pathogen penetration occurred intra and intercellularly and also through the stomata, with intercellular penetration occurring most frequently. The distinct tissues of the petals were colonised, including the vascular system, particularly the xylem. Acervuli were observed on both sides of the petals. Although the fungus did not penetrate through the epidermal cells of the stigma, C. acutatum caused necrosis and an increase of phenolics in this tissue. A protective layer rich in lipophilic and phenolic compounds was formed under the necrotic area and crystals of oxalate were associated with the sites where the pathogen was present.     

结合药液表面张力与苹果树冠层参数预测喷雾药液用量的方法及应用

为了保证果树精准施药,减少药剂流失,通过4种常用表面活性剂调节药液的表面张力,借助界面张力仪、微重力天平和植物冠层扫描仪等测定手段建立了药液表面张力与苹果叶片最大稳定持液量 (R_m) 之间的关系,并结合果树常用冠层参数,提出了一种预估果树施药液量的方法。结果表明:生长前期苹果叶片近轴面的R_m值明显高于生长后期;在同一生长期,苹果叶片远轴面的R_m值均高于近轴面,且R_m值随叶倾角的增大而减小;生长前期苹果叶片远轴面的R_m值受药液表面张力的影响大于近轴面,但随着叶倾角的增大,叶片近、远轴面的R_m受药液表面张力的影响减小。当药液中表面活性剂的浓度未达到其临界胶束浓度 (cmc) 时,药液的表面张力与苹果叶片的R_m值成正相关关系,由此可建立一元线性回归方程,结合果树平均叶片倾角、叶面积指数和冠层地面投影面积等植物冠层参数,可以估算果树的最大施药液用量。采用4种常用的农药制剂验证了所建方法的实用性。该研究结果可以为苹果园喷雾施药时预估药液用量、减少药剂流失提供理论依据。     

Effects of surfactants on primisulfuron activity in barnyardgrass (Echinochloa crus-galli [L.] Beauv.) and green foxtail (Setaria viridis [L.] Beauv.)

Laboratory and greenhouse studies were conducted to measure the contact angle of primisulfuron droplets with and without surfactants on the leaf surfaces of barnyardgrass and green foxtail, to determine the primisulfuron activity on these weed species, and to examine the spray deposit of primisulfuron with and without surfactants on the leaf surface of green foxtail using scanning electron microscopy. A non-ionic surfactant (NIS) and an organosilicone wetting agent (OWA) were used. The contact angles of 1 μL droplets were measured on the leaf surface using a goniometer. The activity of primisulfuron on barnyardgrass and green foxtail was assessed at 3 weeks after treatment based on visual injury and the fresh weight. The contact angles of the droplets of primisulfuron on the adaxial surface of the barnyardgrass and green foxtail leaves were 152° and 127°, respectively, when applied without surfactant. The addition of either surfactant markedly reduced the contact angle for both weed species, which was lowest when the OWA was added to primisulfuron. The percentage injury of barnyardgrass was very low, even at the higher rate of primisulfuron, regardless of the surfactant. Primisulfuron at 40 g ha⁻¹ controlled 43% of green foxtail without surfactant, which increased to 65% with the NIS and 83% with the OWA. Primisulfuron with a surfactant markedly reduced the fresh weight of green foxtail compared with primisulfuron applied alone, regardless of the primisulfuron rate and surfactant type. The scanning electron micrographs showed a uniform deposit of spray droplets, with close contact of the droplets to the leaf epicuticular surface in green foxtail in the presence of a surfactant compared with no surfactant. The enhanced primisulfuron activity on green foxtail with surfactants was related to the reduced contact angle and uniform deposition of the primisulfuron spray droplets on the leaf surface.     

Histology of waxflower (Chamelaucium spp.) flower infection by Botrytis cinerea

Botrytis cinerea infects waxflower (Chamelaucium spp.) flowers and can induce them to abscise from their petioles before disease becomes evident. Botrytis cinerea infection of flowers was studied on two waxflower cultivars by light and electron microscopy. Pot‐grown waxflower flowers were harvested, inoculated with aqueous suspensions of B. cinerea conidia, incubated at 20–22°C and >95% RH and examined within 96 h post‐inoculation (hpi). Conidial germination on petals started 4 hpi, penetration via germ tube tips was 6 hpi and protoappressoria were formed 8 hpi. Germination on petals approximately doubled every 4–6 h to 18 hpi. Conidial germination was ca. 50% at 22–24 hpi. Botrytis cinerea infected most waxflower flower organs, including petals, anthers and filaments, stigma and hypanthium, within 24 hpi. Hyaline and lobate appressoria were observed 36 hpi. Infection cushions on stamen bases were formed 36 hpi by saprophytic hyphae that originated from anthers. This infection process can give rise to tan‐coloured symptoms typical of botrytis disease that radiate from this part of the flower. Subcuticular hyphae were present at high density near stamen bases and evidently resulted from multiple penetrations from single infection cushions. The subcuticular hyphae grew within the hypanthium and towards the centre of the floral tube. When flower abscission occurred, floral tube tissues close to the abscission zone remained uninfected. This observation infers possible transmission of a signal (e.g. ethylene) upon B. cinerea infection. Thus, B. cinerea causes flower abscission apparently as a defence response.     

球毛壳ND35菌株在宿主植物上的侵染定殖

为了解球毛壳Chaetomium globosum ND35菌株在宿主植物上的侵染定殖方式和途径,以毛白杨组培苗为宿主植物,借助光学显微镜、扫描电镜、透射电镜,结合免疫荧光标记技术,研究了球毛壳ND35菌株子囊孢子萌发后在毛白杨上的侵染行为及其菌丝在组培苗根部的定殖。结果显示,子囊孢子萌发后形成的菌丝,能从杨树苗根、茎部表面细胞间的缝隙侵入或在根表面形成附着胞,进而形成侵染钉直接从表皮细胞侵入,在叶部主要从气孔侵入叶片内部。侵入根部的菌丝主要定殖于表皮细胞、外皮层细胞和细胞间隙,未进入内皮层和维管束组织。     

Effect of microclimate and nutrients on development of cucumber gray mold (Botrytis cinerea)

Infection of young parthenocarpic cucumber fruits byBotrytis cinerea begins in the petals. Removing petals or washing nutrients from the flower significantly reduced infection. Germination of conidia occurred at relative humidity (r.h.) above 92%, but when water deposition on artificial surfaces was prevented, germination did not occur even at 98% r.h. Germination of conidia on petals is promoted by deposition of an aqueous film not visible on the petal surface by the bare eye (but demonstrable by CoCl₂). Provided there is a film of water on the surface of the host, germination and the infection process occur at a wide range of temperatures up to 25 °C. Pre-exposure of cucumber plants at temperatures as high as 30 °C or as low as 8 °C, prior to their infection and incubation under conditions conducive to gray mold, resulted in greater severity of the disease on young fruits or leaves as compared with plants previously incubated at 10-25 °C. The relevance of these results to cultural control of gray mold is discussed.     

The role of the abaxial leaf surface waxes of Lolium spp. in resistance to Erysiphe graminis

Field and glasshouse observations of Lolium spp. grasses indicated that the lower, abaxial, leaf surface was rarely infected by powdery mildew ( Erysiphe graminis ) even when the upper, adaxial, surface was densely colonized. Experiments showed that conidia of two strains of E. graminis , one from Lolium and one from Avena , germinated equally well on both surfaces of Lolium and Avena leaves, but that the subsequent growth and development of germlings was impaired on the lower surface of Lolium leaves, so that most formed only multiple short germ tubes or an abnormal long tube, and only c. 25% or fewer formed infection structures. This contributes to the apparent resistance of the lower Lolium leaf surface to powdery mildew and may help to explain why the disease is relatively unimportant in UK ryegrass crops, since infection structures develop at a high frequency on only 50% of the leaf area, i.e. the upper surface. Scanning electron microscopy showed that the epicuticular waxes on the lower Lolium leaf surface form amorphous sheets. This contrasts with the crystalline plate waxes seen on the upper surface of Lolium leaves and on both surfaces of oat leaves. However, when the lower Lolium leaf surface was washed with chloroform to remove epicuticular wax, normal germling and infection structure development was obtained on the wax-free surface. This suggests that the sheet waxes prevent the pathogen gaining access to features of the cuticular membrane which trigger normal germling development.     

Infection of ryegrass by three rust fungi (Puccinia coronata, P. graminis and P. loliina) and some effects of temperature on the establishment of the disease and sporulation

Experiments were conducted to examine the processes leading up to the infection of Lolium temulentum by crown rust ( Puccinia coronata ), stem rust ( P. graminis ) and brown rust ( P. loliina ), and the effects of temperature on these processes and sporulation. Uredia of all three rusts were produced freely if the adaxial leaf surface was inoculated, but did not form following inoculation of the abaxial surface. Light and scanning electron microscopy revealed abnormal growth of germlings on the abaxial surface which had amorphous sheet-like epicuticular waxes and very few stomata. On the adaxial leaf surface germ tubes of all the rusts orientated at right angles to the long axis of the leaf. However, the directional growth of germ tubes was often disrupted when they contacted the surface of bulliform cells at the base of leaf grooves. For P. loliina the optimum temperatures for urediospore germination and sub-stomatal vesicle formation were 12–16°C, and 8–20°C for appressorium formation. The optimum temperatures, for the same stages of fungal development, for P. coronata and P. graminis were higher. Urediospore production of P. loliina was higher at 10°C than at 25°C, but was similar at both temperatures for P. coronata .     

Effects of temperature,vapour pressure deficit and radiation on infectivity of conidia ofBotrytis cinerea and on susceptibility of gerbera petals

The effect of vapour pressure deficit, temperature and radiation on the postharvest susceptibility of gerbera flowers toB. cinerea, on the water relations of gerbera flowers and on the lesion formation after conidial infection ofB. cinerea was studied. The temperature range in whichB. cinerea could germinate and growin vitro is 5–30 °C. In climate chamber experiments flowers had more lesions ofB. cinerea at temperatures of 20 and 25 °C than at 10 and 15°C. At 15, 20 and 25°C the infectivity ofB. cinerea conidia was negatively affected during a storage-period of 7 days. At a vapour pressure deficit (VPD) of 200 Pa significantly more conidia ofB. cinerea were infective than at 800 Pa. At a VPD of 800 Pa the susceptibility of gerbera flowers forB. cinerea was not significantly different than at 200 Pa. High radiation levels in glasshouses in spring and summer negatively influenced the infectivity of conidia ofB. cinerea on the flower surface, but did not affect the susceptibility of gerbera flowers forB. cinerea. In spring and early summer conidia lost their infectivity at high radiation levels, high temperatures and high levels of VPD. In summer gerbera flowers could be more susceptible toB. cinerea because of high temperatures in glasshouses, but the negative effect of radiation on the conidia ofB. cinerea seemed to overrule the temperature effect. Thus, the numbers of lesions in spring and summer can be low compared with the numbers in other seasons, although the numbers ofB. cinerea colonies on spore traps can be high. The effect of temperature on the susceptibility of gerbera flowers can probably be explained by changes of water status in the petals. At higher temperatures the number of lesions and the turgor (=water potential—osmotic potential) in the petals increased. Temperatures <10°C during lesion formation (RH>95% and VPD<50 Pa) had a temporary negative effect on the number of lesions. After 3 days of incubation the numbers of lesions were about equal (30 lesions/cm²) from 5 to 20°C. At 30°C no lesion formation was observed even after 3 days.