Interaction of endodormancy and paradormancy in raspberry (Rubus idaeus L.)

Bud break in protected Northern European raspberry crops is often poor and uneven with many of the sub-apical buds remaining in a dormant state. In order to improve bud break and therefore yields, the mechanism controlling bud dormancy must be determined. Canes of the biennial cultivar ‘Glen Moy’ were forced as isolated single nodes, trisections, or as intact canes after different lengths of cold storage chill unit (CU) accumulation in order to determine whether the buds were in an endodormant or paradormant state. The results showed that buds on the lower parts of the intact canes remained in a dormant state long after buds from higher up the intact cane and also the single nodes from all parts of the cane had emerged from the deepest phase of endodormancy. This would imply that these buds were being held in a paradormant state until large amounts of chilling units (>1000 CU) had been accumulated. The trisected cane portions revealed almost no significant differences in bud break levels throughout the experiment when compared with the single nodes. This suggests that removal of the apical part of the cane would be effective in improving bud break by reducing the paradormant condition. A period of secondary dormancy was also observed in the intact canes which may also exacerbate the poor bud break observed in protected crops. This was not seen in the single nodes or the trisected canes which indicates that treatments which reduce paradormancy may also minimise the risk of secondary dormancy. By identifying the phase of bud dormancy which causes poor bud break, attention can now be focused on methods which overcome paradormancy in protected crops. Such methods might include tipping (removal of the cane apex), horizontal training methods, more efficient chilling methods, and chemical treatments.     

Warm spring temperatures induce persistent season-long changes in shoot development in grapevines

Background and Aims

The influence of temperature on the timing of budbreak in woody perennials is well known, but its effect on subsequent shoot growth and architecture has received little attention because it is understood that growth is determined by current temperature. Seasonal shoot development of grapevines (Vitis vinifera) was evaluated following differences in temperature near budbreak while minimizing the effects of other microclimatic variables.

Methods

Dormant buds and emerging shoots of field-grown grapevines were heated above or cooled below the temperature of ambient buds from before budbreak until individual flowers were visible on inflorescences, at which stage the shoots had four to eight unfolded leaves. Multiple treatments were imposed randomly on individual plants and replicated across plants. Shoot growth and development were monitored during two growing seasons.

Key Results

Higher bud temperatures advanced the date of budbreak and accelerated shoot growth and leaf area development. Differences were due to higher rates of shoot elongation, leaf appearance, leaf-area expansion and axillary-bud outgrowth. Although shoots arising from heated buds grew most vigorously, apical dominance in these shoots was reduced, as their axillary buds broke earlier and gave rise to more vigorous lateral shoots. In contrast, axillary-bud outgrowth was minimal on the slow-growing shoots emerging from buds cooled below ambient. Variation in shoot development persisted or increased during the growing season, well after temperature treatments were terminated and despite an imposed soil water deficit.

Conclusions

The data indicate that bud-level differences in budbreak temperature may lead to marked differences in shoot growth, shoot architecture and leaf-area development that are maintained or amplified during the growing season. Although growth rates commonly are understood to reflect current temperatures, these results demonstrate a persistent effect of early-season temperatures, which should be considered in future growth models.     

高温和单氰胺对油桃休眠花芽呼吸代谢的影响

以3年生盆栽‘曙光’油桃为材料,研究油桃自然休眠过程中50℃高温和单氰胺对花芽呼吸代谢的影响.结果表明:高温和单氰胺均可以打破油桃的自然休眠,导致休眠花芽呼吸代谢显著下降,其呼吸代谢的衰减可持续数小时.主要呼吸途径三羧酸循环(TCA)和磷酸戊糖途径(PPP)的运行均受到影响.未经破眠处理的花芽TCA和PPP均呈衰减趋势,而高温和单氰胺诱导了早期呼吸衰减结束后PPP的迅速活化.高温还表现出对TCA恢复的诱导,而单氰胺在96 h内未表现出这种作用.在高温和单氰胺打破自然休眠的机制中,呼吸衰减和随后出现的PPP活化可能是重要的组成部分.     

Effects of Didymella applanata and Botrytis cinerea on axillary buds, lateral shoots and yield of red raspberry

The viability of axillary buds and the growth and potential yield of lateral shoots at nodes of red raspberry (Rubus idaeus) infected naturally by Didymella applanata or Botrytis cinerea were measured on excised nodes, decapitated nursery canes or on canes from fruiting plantations. In comparison with lesion-free nodes, buds at infected nodes were smaller and fewer of them were capable of growth when excised and ‘forced’, although the difference in growth between infected and uninfected nodes decreased during late winter. After February, those buds at infected nodes which were capable of forced growth did so as early and with a similar growth rate as those at lesion-free nodes. In April, 70% of buds at infected nodes were capable of growth compared with 94% of those at lesion-free nodes. When nursery canes of cv. Mailing Delight were decapitated above infected nodes the emergence of lateral shoots from the terminal infected node did not differ significantly from that at lesion-free nodes. On a range of farm sites in Scotland the emergence of shoots at infected nodes in the cropping region of canes was significantly poorer than from uninfected nodes but substantially better at infected nodes of cv. Glen Clova than at those of cvs Mailing Jewel and Mailing Orion. It is suggested that cv. Glen Clova is relatively tolerant of spur blight and cane botrytis. The length and potential yield of laterals which developed at infected nodes in the cropping region of canes in these three cultivars did not differ significantly from those at lesion-free nodes. In all tests there was no significant difference in growth at nodes infected by D. applanata and B. cinerea which may indicate a common mechanism for suppression of buds.     

Expression analysis of phytochromes A, B and floral integrator genes during the entry and exit of grapevine-buds from endodormancy

A common molecular regulatory pathway that involves PHYA, PHYB and floral integrator genes CONSTANS (CO), FLOWERING LOCUS T (FT) and SUPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) has been suggested to participate in the regulation of photoperiod dependent processes such as flowering and dormancy. In grapevines (Vitis vinifera L.), decreasing photoperiod and low temperatures trigger the transition of buds into endodormancy (ED), a process that is accompanied by drastic changes in gene expression of VvPHYA and B in leaves. To analyse the relationship of VvPHYA, VvPHYB, and grape homologues of Arabidopsis floral integrator genes VvCO, VvFT, VvMADS8, with ED, a comparative expression analysis of these genes was performed in grapevine-leaves and buds before, during and after the transition of buds into ED. The expression of all the above genes in the bud-tissue, and the fact that photoperiod regulates differently the expression of VvPHYA and B in buds than in leaves, suggests that the bud might be an autonomous or semi-autonomous organ in perceiving and transducing the photoperiod signal. On the other hand, the coordinated down-regulation of VvFT in leaves and buds during the transition of buds into ED, and its subsequent up-regulation following the application of dormancy-breaking compounds, hydrogen cyanamide (HC) and sodium azide, suggests that VvFT could play a key role in stimulating bud-growth by repressing their entry into ED.     

甘蔗双腋芽突变体表型鉴定及应用潜力评估

蔗茎腋芽是甘蔗无性繁殖的主要器官,充足的下芽量保证了甘蔗的高产稳产,但同时也造成用种量大、种植成本高,是生产上一直难以平衡的问题。本研究以大茎野生种创新利用过程中获得的1份蔗茎孪生双腋芽的突变体种质云蔗07-86为材料,对其双腋芽表型、稳定性、可遗传性及对产量的影响进行鉴定和测试。研究发现该突变体的双腋芽以孪生、邻接和分离3种方式随机着生于蔗茎中上部,通常从地上第6茎节开始发生双腋芽,发生频率自下而上呈现从无到有、先增后减的总体趋势,且播种后2个腋芽均能正常萌发;通过连续多年的无性繁殖大田试验观察发现,双腋芽性状能够在繁殖群体中稳定保持;利用该突变体与32份不同的甘蔗种质进行杂交,仅在11个组合中的极少数杂交后代无性系上观察到双腋芽现象,表明双腋芽性状可以通过杂交传递给后代,但双腋芽在后代群体中出现的概率极低;以相同下芽量分别将突变体单、双腋芽种茎进行种植,发现不同腋芽表型类型的有效茎数、蔗茎产量差异不显著,表明双腋芽突变体具备保证下芽量而降低用种量的应用潜力。本研究为后期开展双腋芽形成候选基因挖掘及双腋芽突变育种利用奠定基础。     

THE FATE OF THE DWARF SHOOT APEX IN BRISTLECONE PINE (PINUS LONGAEVA)

The fate of the pine dwarf shoot (DS) apex after needle initiation has been controversial. Dwarf shoot primordia of Pinus longaeva were examined to determine the developmental basis for DS with and without interfoliar buds. Interfoliar buds are microscopic buds derived from the original terminal apex of the DS. In October, all the DS primordia are similar in size and appearance. However, as the needles elongate in the following June the apices of more proximal DS decrease in size, such that by July there is a clear diminishing size gradient of apical domes in going from the most distal to the most proximal positions. The distal DSs start to form bud scales in July and have fully formed interfoliar buds by mid-August. In contrast, those DS apices lacking protective bud scales at needle maturity become suberized and can never proliferate into long shoots. The distal placement of interfoliar buds may be due to a group effect, where each developing DS inhibits the more proximal DSs in the long shoot terminal bud.     

Dormant Buds and Adventitious Root Formation by Vitis and Other Woody Plants

Viticulture has historically depended upon clonal propagation of winegrape, tablegrape, and rootstock cultivars. Dependence on clonal propagation is perpetuated by consumer preference, legal regulations, a reproductive biology that is incompatible with sustaining genetic lines, and the fact that grapevine breeding is a slow process. Adventitious root formation is a key component to successful clonal propagation. In spite of this fact, grapevine has not been a centerpiece for adventitious root research. Dormant woody canes represent complex assemblages of tissues and organs. Factors that further contribute to such complexity include levels of endogenous plant growth regulators, the extent and duration of dormancy, carbohydrate storage, transport, the presence or absence of dormant buds or emergent shoots, and preconditioning treatments. For the above reasons, the mechanisms driving adventitious root formation by grapevine and other woody cuttings are poorly understood. We present results indicating that the dormant bud on cane cuttings from a non-recalcitrant to root Vitis vinifera cultivar, cv. Cabernet Sauvignon, slows or inhibits adventitious root emergence. In contrast to Cabernet Sauvignon, removal of the dormant bud from cane cuttings of a recalcitrant to root hybrid rootstock (V. berlandieri × V. riparia cv. 420A) and an intermediate to root hybrid rootstock (V. riparia × V. rupestris cv. 101-14) had no influence on adventitious root emergence. Reciprocal transplanting of nodes containing dormant buds among all three cultivars did not affect rooting behavior. Our results indicate that the commonly held belief that bud removal diminishes adventitious root emergence is not true.     

Dormant Buds and Adventitious Root Formation by <Emphasis Type="Italic">Vitis</Emphasis> and Other Woody Plants

Viticulture has historically depended upon clonal propagation of winegrape, tablegrape, and rootstock cultivars. Dependence on clonal propagation is perpetuated by consumer preference, legal regulations, a reproductive biology that is incompatible with sustaining genetic lines, and the fact that grapevine breeding is a slow process. Adventitious root formation is a key component to successful clonal propagation. In spite of this fact, grapevine has not been a centerpiece for adventitious root research. Dormant woody canes represent complex assemblages of tissues and organs. Factors that further contribute to such complexity include levels of endogenous plant growth regulators, the extent and duration of dormancy, carbohydrate storage, transport, the presence or absence of dormant buds or emergent shoots, and preconditioning treatments. For the above reasons, the mechanisms driving adventitious root formation by grapevine and other woody cuttings are poorly understood. We present results indicating that the dormant bud on cane cuttings from a non-recalcitrant to root Vitis vinifera cultivar, cv. Cabernet Sauvignon, slows or inhibits adventitious root emergence. In contrast to Cabernet Sauvignon, removal of the dormant bud from cane cuttings of a recalcitrant to root hybrid rootstock (V. berlandieri × V. riparia cv. 420A) and an intermediate to root hybrid rootstock (V. riparia × V. rupestris cv. 101-14) had no influence on adventitious root emergence. Reciprocal transplanting of nodes containing dormant buds among all three cultivars did not affect rooting behavior. Our results indicate that the commonly held belief that bud removal diminishes adventitious root emergence is not true.     

Effects of hydrogen cyanamide on antioxidant enzymes’ activity,proline and polyamine contents during bud dormancy release in Superior Seedless grapevine buds

The effect of hydrogen cyanamide (HC) on dormancy release, antioxidant enzyme’s activity and proline and free polyamine contents were investigated in ‘Superior Seedless’ grapevine buds. HC application caused a sharp decrease of catalase (CAT, EC 1.11.1.6) activity and a transient stimulation during the 5 days following treatment of peroxidase (POD, EC 1.11.1.7) and ascorbate peroxidase (APX, EC 1.11.1.11) activities. This coincided with an accumulation of total free polyamines, especially putrescine (Put). Proline content increased dramatically. There was a strong correlation between APX and POD activities and total free PAs and Put contents implying a possible stimulating effect of the latter compounds on these enzymes. These observations indicate that HC triggers an oxidative stress leading to bud endodormancy release. Afterward, as budbreak started, we observed a rapid proline and Put degradation; this could be responsible for reactivation of growth. Indeed, the decline in Put to (Spd + Spm) ratio, reported here, may be considered as a reliable biochemical marker of bud growth resumption.     

MORPHOLOGICAL AND ULTRASTRUCTURAL DEVELOPMENT AND STARCH ACCUMULATION DURING CHILLING OF SOUR CHERRY FLOWER BUDS

Floral tissue of dormant sour cherry (Prunus cerasus L.) flower buds was examined by light and electron microscopy during field development and laboratory chilling. Differentiation of flower parts ceased by early November and there was no further morphological development until visible signs of budbreak occurred after the end of rest. Pollen meiosis occurred at the half green budbreak stage. No ultrastructural changes accompanied chilling accumulation except development of numerous starch grains in plastids. Buds on cut shoots held at the non-chilling temperature of 15 C developed more mitochondria but acquired no starch when compared with buds on cut shoots held at a 5 C chilling temperature. Nuclei and nucleoli enlarged with chilling in ovules and sporogenous cells but not in ovary parenchyma. Accumulation of starch with chilling in most bud tissues was demonstrated histochemically.     

Can phenological models predict tree phenology accurately in the future? The unrevealed hurdle of endodormancy break

The onset of the growing season of trees has been earlier by 2.3 days per decade during the last 40 years in temperate Europe because of global warming. The effect of temperature on plant phenology is, however, not linear because temperature has a dual effect on bud development. On one hand, low temperatures are necessary to break bud endodormancy, and, on the other hand, higher temperatures are necessary to promote bud cell growth afterward. Different process‐based models have been developed in the last decades to predict the date of budbreak of woody species. They predict that global warming should delay or compromise endodormancy break at the species equatorward range limits leading to a delay or even impossibility to flower or set new leaves. These models are classically parameterized with flowering or budbreak dates only, with no information on the endodormancy break date because this information is very scarce. Here, we evaluated the efficiency of a set of phenological models to accurately predict the endodormancy break dates of three fruit trees. Our results show that models calibrated solely with budbreak dates usually do not accurately predict the endodormancy break date. Providing endodormancy break date for the model parameterization results in much more accurate prediction of this latter, with, however, a higher error than that on budbreak dates. Most importantly, we show that models not calibrated with endodormancy break dates can generate large discrepancies in forecasted budbreak dates when using climate scenarios as compared to models calibrated with endodormancy break dates. This discrepancy increases with mean annual temperature and is therefore the strongest after 2050 in the southernmost regions. Our results claim for the urgent need of massive measurements of endodormancy break dates in forest and fruit trees to yield more robust projections of phenological changes in a near future.     

Preformation and neoformation in shoots of Nothofagus antarctica (G. Forster) Oerst. (Nothofagaceae) shrubs from northern Patagonia

The size (length and diameter) and number of leaf primordia of winter buds of Nothofagus antarctica (G. Forster) Oerst. shrubs were compared with the size and number of leaves of shoots derived from buds in equivalent positions. Buds developed in two successive years were compared in terms of size and number of leaf primordia. Bud size and the number of leaf primordia per bud were greater for distal than for proximally positioned buds. Shoots that developed in the five positions closest to the distal end of their parent shoots had significantly more leaves than more proximally positioned shoots of the same parent shoots. The positive relationship between the size of a shoot and that of its parent shoot was stronger for proximal than for distal positions on the parent shoots. For each bud position on the parent shoots there were differences in the number of leaf primordia per bud between consecutive years. The correlations between the number of leaf primordia per bud and bud size, bud position and parent shoot size varied between years. Only shoots produced close to the distal end of a parent shoot developed neoformed leaves; more proximal sibling shoots consisted entirely of preformed leaves. Leaf neoformation, a process usually linked with high shoot vigour in woody plants, seems to be widespread among the relatively small shoots developed in N. antarctica shrubs, which may relate to the species' opportunistic response to disturbance.     

甘蔗健康种子发芽率影响因素分析

该研究主要通过温室沙培试验,分析了甘蔗健康种子单芽材料各因素(单芽长度、不同芽位、泡水时间)及甘蔗基因型、种衣剂及保存天数三种因素对甘蔗健康种子发芽率的影响。结果表明:不同浸泡时间、不同单芽长度和芽位对健康种子单芽发芽率的影响极显著。随着单芽长度的增加,发芽率有明显的提高;随着泡水时间的增加,发芽率不断降低,但各处理之间差异不显著;顶部芽位的发芽率较高,但其他芽位之间发芽率的差异不明显。甘蔗健康种子发芽率具有明显的基因型差异,其中GT28和ROC22的发芽率较高,发芽率在70%左右。种衣剂包衣处可显著提高甘蔗健康种子的发芽率,与其他种衣剂相比扑力猛包衣处理的甘蔗健康种子发芽率最高,且差异显著。随着保存天数的增加,甘蔗健康种子的发芽率不断下降,且差异显著,当保存时间超过8 d时,发芽率低于60%。     

Is GABA-shunt functional in endodormant grapevine buds under respiratory stress?

It has been suggested that a respiratory stress is part of the mechanism through which the dormancy-breaking compounds, hydrogen cyanamide (HC) and sodium azide, induce the release of buds from the endodormancy (ED) in grapevines. The accumulation of metabolites like succinate, alanine (Ala) and γ-amino butyric acid (GABA), together with the activation of the GABA-shunt pathway, is a general feature of plants in response to oxygen deprivation and to respiratory stress. Unexpectedly, in a previous study, we found that GABA applied exogenously to grapevine buds, down-regulated the expression of most genes encoding for antioxidant enzymes, suggesting that its accumulation under respiratory stress conditions could be deleterious for the bud. In order to analyze whether GABA accumulates under respiratory stress conditions in grapevine buds, we analysed in this study, the effect of hypoxia, the respiration inhibitor KCN and the dormancy breaker compound HC, on the level of GABA, and on the expression levels of the GABA-shunt genes (VvGAD, VvGABA-T, VvSSADH). Additionally, genes from the Ala fermentative pathway (VvAlaAT, VvAspAT) were also analysed. The results revealed that although the three treatments mentioned above, up-regulated the expression of VvGAD1, the content of GABA remained constant, while Ala content increased. The lack of GABA accumulation under respiratory stress is an important physiological fact in grapevine buds, since it avoids the down-regulation of antioxidant genes, and promotes the incorporation of succinate into the TCA cycle, a fact that would be important in the release of buds from the ED.     

Expressional regulation of PpDAM5 and PpDAM6, peach (Prunus persica) dormancy-associated MADS-box genes, by low temperature and dormancy-breaking reagent treatment

The present study investigated the expressional regulation of PpDAM5 and PpDAM6, two of the six peach (Prunus persica) dormancy-associated MADS-box genes, in relation to lateral bud endodormancy. PpDAM5 and PpDAM6 were originally identified as homologues of Arabidopsis SHORT VEGETATIVE PHASE/AGAMOUS-LIKE 24 identified in the EVERGROWING locus of peach. Furthermore, PpDAM5 and PpDAM6 have recently been suggested to be involved in terminal bud dormancy. In this study, seasonal expression analyses using leaves, stems, and lateral buds of high-chill and low-chill peaches in field conditions indicated that both genes were up-regulated during the endodormancy period and down-regulated with endodormancy release. Controlled environment experiments showed that the expression of both PpDAM5 and PpDAM6 were up-regulated by ambient cool temperatures in autumn, while they were down-regulated by the prolonged period of cold temperatures in winter. A negative correlation between expression levels of PpDAM5 and PpDAM6 and bud burst percentage was found in the prolonged cold temperature treatment. Application of the dormancy-breaking reagent cyanamide to endo/ecodormant lateral buds induced early bud break and down-regulation of PpDAM5 and PpDAM6 expression at the same time. These results collectively suggest that PpDAM5 and PpDAM6 may function in the chilling requirement of peach lateral buds through growth-inhibiting functions for bud break.     

Possible role of catalase in post-dormancy bud break in grapevines

Changes in the activity of catalase (Cat) and in the levels of H2O2 were followed throughout dormancy in buds of grapevines (Vitis vinifera L.). In grapevines grown in the Elqui valley in Chile, a region with warm-winters, the activity of Cat increased during the recess period of buds, reaching a maximum and thereafter decreased to less than one third of its maximal activity. Three isoforms of Cat were detected in extracts of buds by native PAGE analysis, and the extracted activity was inhibited competitively by hydrogen cyanamide (HC), a potent bud-break agent. Furthermore, HC applications to field-grown grapevines in addition to the expected effect on advancing bud break, reduced the Cat activity during bud dormancy. Similar reductions were observed during dormancy in buds of grapevines grown in the Central valley in Chile, a region with temperate winters, suggesting that HC and winter chilling inhibits the activity of the main H2O2 degrading enzyme in grape buds. A transient rise in H2O2 levels preceded the release of buds from endodormancy, moreover, the peak of H2O2 and the onset of bud break occurred earlier in HC treated than in control grapevines, suggesting the participation of H2O2 as a signal molecule in the release of endodormancy in grape buds. The relationship between Cat inhibition, rise in H2O2 levels and initiation of bud break are discussed.     

Axillary bud banks of two semiarid perennial grasses: occurrence, longevity, and contribution to population persistence

The occurrence, longevity, and contribution of axillary bud banks to population maintenance were investigated in a late-seral perennial grass, Bouteloua curtipendula, and a mid-seral perennial grass, Hilaria belangeri, in a semiarid oak-juniper savanna. Axillary buds of both species were evaluated over a 2-year period in communities with contrasting histories of grazing by domestic herbivores. A double staining procedure utilizing triphenyl tetrazolium chloride and Evan's blue indicated that both viable and dormant axillary buds remained attached to the base of reproductive parental tillers for 18–24 months which exceeded parental tiller longevity by approximately 12 months. Bud longevity of the late-seral species, B. curtipendula, exceeded bud longevity of the mid-seral species, H. belangeri, by approximately 6 months. Younger buds located on the distal portion of the tiller base were 3.2 and 1.4 times more likely to grow out than older proximal buds of B. curtipendula and H. belangeri, respectively. The percentage of older proximal buds, which included comparable portions of viable and dormant buds, that grew out to produce tillers following mortality of parental tillers was 6.0% for B. curtipendula and 8.4% for H. belangeri. In spite of the occurrence of relative large axillary bud banks for both species, the magnitude of proximal bud growth did not appear sufficient to maintain viable tiller populations. We found no evidence to support the hypothesis of compensatory bud growth on an individual tiller basis for either species. Grazing history of the communities from which the buds were collected did not substantially affect the number, status, longevity, or outgrowth of axillary buds on an individual tiller basis for either species. However, long-term grazing by domestic herbivores influenced axillary bud availability by modifying population structure of these two species. Bud number per square meter for B. curtipendula was 25% lower in the long-term grazed compared to the long-term ungrazed community based on a reduction in both tiller number per plant and plant number per square meter. In contrast, bud number per square meter for H. belangeri was 190% greater in the long-term grazed than in the long-term ungrazed community based on a large increase in plant density per square meter. Minimal contributions of axillary bud banks to annual maintenance of tiller populations in this mid- and late-seral species underscores the ecological importance of consistent tiller recruitment from recently developed axillary buds. Consistent tiller recruitment in grasslands and savannas characterized by intensive grazing and periodic drought implies that (1) bud differentiation and maturation must be remarkably tolerant of adverse environmental conditions and/or (2) tiller recruitment may resume from buds that mature following the cessation of severe drought and/or grazing, rather than from mature buds that survive these disturbances. These scenarios warrant additional research emphasis given the critical importance of this demographic process to tiller replacement in species populations and the maintenance of relative species abundance in grasslands and savannas. Received: 12 August 1996 / Accepted: 30 December 1996     

Colonization of Japanese beech leaves by phyllosphere fungi

To examine the colonization of living leaves from buds and twigs by phyllosphere fungi of Japanese beech, the mycobiota were investigated on buds and twigs and on leaves covered with well-ventilated bags before budbreak. Ten phyllosphere fungi were isolated from rolled-up leaves within buds, bud scales, and twigs. However, frequencies of phyllosphere fungi on expanded leaves were reduced markedly when the buds were covered with well-ventilated bags before budbreak compared to the leaves that were not covered. This observation suggests that invasion of the fungi to the leaves from buds and twigs may be possible but is not the main route. Horizontal transmission may be common in endophytes and epiphytes of beech leaves. Phyllosphere mycobiota were then compared between sun and shade leaves. Of 13 species recorded as phyllosphere fungi, the frequencies of 2 species were lower and those of 3 species were higher in sun leaves than in shade leaves. Frequencies of the other 8 phyllosphere species were not different between sun and shade leaves. This result indicates that the colonization of leaves by some phyllosphere fungi was affected by the microenvironmental conditions on leaf surfaces.