水稻纹枯病菌营养及寄主资源生态位

由于IPM概念的局限性,有害生物生态调控（EPM）理论和方法的提出发展了IPM,生态位原则是有害生物生态调控（EPM）的重要原则之一,生态位研究为EPM的具体实施提供了依据。应用可持续农业和EPM理论及生态位理论研究了水稻纹枯病的生态位,分析了水稻纹枯病菌氮肥营养生态位和寄主品种资源生态位,结果表明：以相对侵染效率作为指标,在水稻不同生育期,纹枯病的氮肥营养生态位宽度不同,其中以孕穗期的生态位宽度最小,为0.6979,拔节期、抽穗期、灌浆期和乳熟期的生态位宽度分别是0.9741,0.8884,0.7974和0.9815,表明水稻纹枯病在水稻不同生育阶段利用氮肥的效能不同。寄主品种资源生态位宽度在拔节期、孕穗期、抽穗期、乳熟期分别为0.9348,0.7677,0.8875和0.9962。以病情指数为指标,氮肥营养生态位宽度在拔节期、孕穗期、抽穗期、灌浆期和乳熟期分别为0.9379,0.9696,0.6775,0.6729和0.7691。其氮肥营养生态位宽度在拔节期与孕穗期最大,生态位宽度指数接近于1。寄主品种资源生态位宽度在各生育期均接近1,表明寄主品种资源生态位宽度在各生育期是相似的,即说明水长期稻纹枯病菌利用品种资源各状态的选择和利用效能是相似的。     

Cloning and overexpression of antifungal barley chitinase gene in Escherichia coli

Plant chitinases are pathogenesis-related proteins, which are believed to be involved in plant defense responses to pathogen infection. In this study, chitinase gene from barley was cloned and overexpressed in Escherichia coli. Chitinase (35 kDa) was isolated and purified. Since the protein was produced as insoluble inclusion bodies, the protein was solubilized and refolded. Purified chitinase exerted broad-spectrum antifungal activity against Botrytis cinerea (blight of tobacco), Pestalotia theae (leaf spot of tea), Bipolaris oryzae (brown spot of rice), Alternaria sp. (grain discoloration of rice), Curvularia lunata (leaf spot of clover) and Rhizoctonia solani (sheath blight of rice). Due to the potential of broad-spectrum antifungal activity barley chitinase gene can be used to enhance fungal-resistance in crop plants such as rice, tobacco, tea and clover.     

Immunology of the pathogen virulence and phytotoxin production in relation to disease severity: a case study in sheath blight of rice

Polyclonal antibodies against purifiedRhizoctonia solani toxin obtained from infected rice sheath tissues (sheath blight toxin, SBT) and culture filtrates (culture filtrate toxin, CFT) were developed in rabbit and chicken. The IgG was isolated from serum and egg yolk of rabbit and chicken, respectively, and their specificity was investigated by indirect ELISA. Antibodies developed against CFT and SBT in rabbits exhibited relatively higher titer values when compared to chicken antibodies. Positive correlation was observed between the degree of sheath blighting and the levels of antigens induced by each isolate during sheath blight symptome development as detected by rabbit SBT antibody and the isolate RS7 was identified as most virulent. Optimization of incubation period for maximum toxin production in liquid medium and rice sheaths indicated that the production of CFT and SBT is maximum after 15 d and 6 d of pathogen inoculation. Studies of the possible translocation of RS-toxin in rice plants upon inoculation withR. solani showed downward translocation as detected by rabbit/chicken SBT antibodies. Since plant inoculation required a higher concentration of inoculum and maintenance of plants, serological assay by ELISA is more sensitive than whole-plant assays in detecting RS-toxin, with the advantage that ELISA also allows rapid determination of RS-toxin production.     

水稻施硅对白背飞虱刺吸和寄主选择行为的影响

【目的】硅可增强植物对多种植食性昆虫的抗性。本研究旨在了解硅处理对水稻叶鞘硅化程度及其对白背飞虱Sogatella furcifera刺吸和寄主选择行为的影响,以明确施硅增强水稻对白背飞虱的抗性的部分机制。【方法】采用扫描电镜观察施硅水稻的叶鞘硅化程度;利用刺吸电位技术记录白背飞虱若虫刺吸行为;采用笼罩法测定白背飞虱雌成虫栖息和产卵选择性。【结果】与不施硅的对照相比,硅处理(0.16和0.32 g SiO₂/kg土壤)增加了水稻叶鞘硅细胞的数量,延长了白背飞虱若虫刺吸行为中非刺探波和路径波的总时间,缩短了取食韧皮部汁液的时间。在选择性测定中,与对照组相比,白背飞虱雌成虫对0.16和0.32 g SiO₂/kg土壤处理的水稻的栖息率分别降低48.0%和67.4%,在其上的产卵量分别降低34.8%和46.1%。【结论】施硅增加水稻对白背飞虱的排驱性,阻碍白背飞虱的刺吸行为,因此有助于增强水稻对白背飞虱的抗性。     

Detection and characterization of nitrogen circulation through the sieve tubes and xylem vessels of rice plants

Nitrogen movement through the xylem vessels and sieve tubes in rice plants was studied using xylem and phloem sap analysis in combination with stable and radioactive nitrogen isotope techniques.More than 90% of nitrogen was translocated in the sieve tubes of rice plants as amino acids. When 15N (99.6 atom%) was applied as a nitrate to the root, 15N first appeared in phloem sap of the leaf sheath within 10 minutes and increased to 37 atom% excess 5 hours after the experiment had started. In long-term experiments, 63% of nitrogen in the phloem sap of the leaf sheath and 15% in that of the uppermost internode came from nitrogen absorbed within the last 24 hours and 50 hours, respectively.To obtain information about the more rapid circulation of nitrogen in the plant, radioactive 13N was used as a tracer. A positron-emitting tracer imaging system was used to show that 13N was transferred to the leaf sheath within 8 minutes of its application to the roots. Analysis of the xylem sap of the leaf sheath showed that when the nitrate was applied to the roots, most of the nitrogen in the xylem was transported as a nitrate.These data showed that phloem and xylem sap analysis together with the stable and radioactive nitrogen techniques provide a good method for the detection of nitrogen cycles in plants.     

Agrobacterium-mediated transformation of indica rice with chitinase gene for enhanced sheath blight resistance

Four rice indica genotypes of local importance were transformed with RC7, rice chitinase cDNA clone through Agrobacterium-mediated gene transfer method using mature seed derived calli as explants. The putative hygromycin resistant calli showed varied level of regeneration efficiency ranging from 2.0 to 7.6 %. The stable integration and expression of RC7 was confirmed through polymerase chain reaction (PCR) and Western analysis. Transformation efficiency ranged from 0.9 to 5.2 %. The expression of RC7 (35 kDa chitinase) in different tissues of transgenic plant (root, sheath and leaf) was proved through Western analysis and in terms of increased chitinase activity. The inheritance of transgene was studied through PCR and Western analysis in transgenic plants of Pusa Basmati 1. Bioassays with transgenic plants of local cultivars exhibited enhanced resistance up to 33.3 % to rice sheath blight pathogen Rhizoctonia solani under glasshouse conditions. Enhanced expression or 3-to 4-fold increased activity of chitinase in transgenic plants was correlated with sheath blight resistance.     

Symptomology and etiology of a new disease,yellow stunt,and root rot of standing milkvetch caused by <Emphasis Type="Italic">Embellisia</Emphasis> sp. in Northern China

An Embellisia sp. has been established as the cause of a new disease of the herbaceous perennial forage legume, ‹standing milkvetch’ (Astragalus adsurgens Pall.) in Northern China, which severely reduces plant density and degrades A. adsurgens stands. The disease was common at an experimental location in Gansu Province where it was recognized by the occurrence of stunted plants with reddish-brown stems and yellow and necrotic leaf blades. An Embellisia sp. was isolated from symptomatic stem, leaf blade, petiole, and root tissues at varying frequencies of up to 90%. Single-spore isolates grew very slowly on PCA, PDA, V-8 and, wheat hay decoction agar. Pathogenicity was confirmed by inoculation of seeds, dipping 2-day-old pre-germinated seedlings in inoculum and spraying inoculum on 6-month-old plants. Symptoms on test plants included yellow leaf lesions, brown lesions on stems and petioles, stunted side-shoots with yellow, small, distorted and necrotic leaves, shoot blight, bud death, crown rot, root rot, and plant death. The disease is named as ‹yellow stunt and root rot’ of A. adsurgens to distinguish it from diseases caused by other known pathogens. Embellisia sp. is also pathogenic to A. sinicus but not to 11 other tested plant species.     

稻麦叶片氮积累量与冠层反射光谱的定量关系

作物氮素积累动态是评价作物群体长势及估测产量和品质的重要指标,对于作物氮素的实时监测和精确管理具有重要意义。该文以5个小麦(Triticum aestivum)品种和3个水稻(Oryza sativa)品种在不同施氮水平下的3年田间试验为基础,综合研究了稻麦叶片氮积累量与冠层反射光谱的定量关系。结果表明,不同试验中拔节后叶片氮积累量均随施氮水平呈上升趋势;稻麦冠层光谱反射率在不同施氮水平下存在明显差异,可见光区(460~710 nm)反射率一般随施氮水平的增加逐渐降低,近红外波段(760~1 220 nm)反射率却随施氮水平的增加逐渐升高;就单波段而言,810和870 nm处的冠层光谱反射率均与稻麦叶片氮积累量具有相对较高的相关性;在光谱参数中,比值植被指数(Ratio vegetation index, RVI)(870,660)和RVI(810,660)均与稻麦叶片氮积累量具有高度的相关性,且相关系数明显高于单波段反射率,尤其是水稻作物;对于小麦和水稻,均可以利用统一的波段和光谱指数来监测其叶片氮积累量,并可以采用统一的回归方程来描述其叶片氮积累量随单波段反射率和反射光谱参数的变化模式,但若采用单独的回归系数则可以提高稻麦叶片氮积累量估测的准确性。     

Evolutionarily stable leaf area production in plant populations

Using an analytical model, it was shown that for a given amount of nitrogen in the canopy of a stand (N(T)), there exists an evolutionarily stable leaf area index (ES-LAI), and therefore an evolutionarily stable average leaf nitrogen content (n(ES)(av);n(ES)(av) =N(T)/ES-LAI), at which no individual plant in the stand can increase its photosynthesis by changing its leaf area. It was also shown that this ES-LAI is always greater than the optimal LAI that maximizes photosynthesis per unit N(T) of the stand. This illustrates that the canopy structure that maximizes photosynthesis of a population is not the same as the canopy structure that maximizes photosynthesis of individuals within a population. It was further derived that the ES-LAI at given N(T) increases with the ratio between the light-saturated photosynthesis and the N content per unit leaf area (leaf-PPNUE) and that it decreases with the canopy extinction coefficient for light (K(L)), the light availability and the apparent quantum yield (phi). These hypotheses were tested by comparing calculated ES-LAI and n(ES)(av) values to actual LAIs and leaf N contents measured for stands of a large variety of herbaceous plants. There was a close correspondence between the calculated and measured values. As predicted by the model, plants with high leaf-PPNUEs produced more leaf area per unit nitrogen than those with low leaf-PPNUEs while plants with horizontal leaves, forming stands with higher K(L) values, produced less leaf area than those with more vertically inclined leaves. These results suggest that maximization of individual plant photosynthesis per unit of nitrogen plays an important role in determining leaf area production of plants and the resulting canopy structure of stands of vegetation. They further suggest this optimization to be a mechanism by which leaf traits such as leaf-PPNUE and leaf inclination angle are causally related to structural characteristics of the population, i.e. the leaf area index of the stand.     

Lesion and epidemic development of Alternaria longipes (Ell. & Ev.) Mason on tobacco

Lesion development of A. longipes was studied in the glasshouse and in the field. The incubation period on glasshouse plants varied from 3 to 8 days. Some pinpoint lesions failed to develop, and lesion development on the upper leaves of pot plants was less than on the lower ones. The maximum estimated lesion multiplication rate was 2.1 daughter lesions/mother lesion/day, but was generally ≤ 0.6. Epidemic development was studied in four seasons (1966 to 1969) by weekly assessment of infection levels on alternate leaves. Leaf area, airborne inoculum concentrations, air, leaf and soil temperature, soil moisture, saturation deficit, rainfall, duration of leaf wetness, and wind speed were also measured. Relative infection rates varied from 0.17-0.26 units/day. Slow disease development in 1968 (r = 0.17 units/day) could be related to the adverse effects of climate on host development and on infection processes. The relative rate of infection varied widely at different stalk position (0.16-0.29 units/day in 1966) and at different times at the same stalk position (0.17=0.48 units/day at position 25 in 1969). There was no consistent relationship between the stalk position of leaves and the rate at which they were infected. Similarly there was no consistent relationship between the rate of infection of whole plants or of leaves at a particular stalk position and climatic or inoculum factors, although during limited periods, changes in one or other of these factors did coincide with an alteration in the relative rate of infection. The interpretation of rates of infection and the theoretical results of greater field hygiene are discussed.     

水稻冠层光合有效辐射的时空分布特征

以2个不同株型水稻品种为材料,设置高、中、低3个施氮水平,利用SunScan冠层分析仪于灌浆期系统测定了不同施氮水平下不同株型水稻品种植株形态和冠层内光合有效辐射（PAR）的时空分布状况.结果表明：施氮量对水稻株高、穗弯曲度和茎叶夹角有明显影响;群体叶面积的垂直分布呈中部>上部>下部的分布特征,最大分层叶面积指数（LAI）出现在0.60相对高度处.冠层内平均PAR透光率从顶部向下递减,且在冠层上中部递减迅速,下部递减缓慢;平均PAR透光率随施氮量的增加而递减;平均PAR透光率日变化表现为早晚较低,中午较高;平均PAR透光率随向下累积LAI的增加呈指数递减,群体消光系数K的日变化表现为早晚较高,中午较低,灌浆期的K值介于0.35～0.50.水稻冠层内PAR的三维空间分布表现为冠层上中部水平面上PAR透光率高,光斑面积大;下部水平面上PAR透光率低,光斑少;同一冠层高度水平面上的PAR光强呈不均匀分布.株型紧凑的水稻品种,冠层透光率高,透光率日变化大,群体消光系数小.     

Effects of inoculum concentration, leaf age and wetness period on the development of dark leaf and pod spot (Alternaria brassicae) on oilseed rape (Brassica napus)

Experiments were done under controlled environment and glasshouse conditions to study the effects of inoculum concentration, leaf age and wetness period on the development of dark leaf and pod spot (Alternaria brussicae) on oilseed rape (Brassica napus). On leaves of potted oilseed rape plants (cv. Bienvenu) inoculated with A. brassicae conidial suspensions, the severity (number of lesions cm^-2) of dark leaf spot increased as inoculum concentration increased from 80 to 660 spores ml^-1and as leaf age increased from 4 to 14 days. On pods on detached racemes of spring oilseed rape (cv. Starlight), the incidence of dark pod spot (% of pods diseased) increased as inoculum concentration increased from 80 to 10⁴spores ml^-1. Increasing inoculum concentration above 10⁴spores ml^-1did not increase the incidence but did increase the severity of dark pod spot. A minimum wetness period of 4 h was needed for infection of oilseed rape leaves (cv. Envol) by A. brussicue at 18°C and disease severity increased with increasing wetness period up to 12 h. The length of dry interruptions after 3–8 h of initial wetness affected the severity of dark leaf spot. A second wetness period increased the severity of dark leaf spot if the dry interruption was ≤ 6 h and if the first wetness period was ≤ 8 h. The incubation period of A. brassicae decreased from 3.5 to 2.5 days as inoculum concentration increased from 80 to 660 spores ml^-on leaves (cv. Bienvenu) at 17–25°C and from 3.8 to 1.0 day as inoculum concentration increased from 80 to ≥2 ≥ 10³spores ml^-1on pods (cv. Starlight) at 18°C.     

Rice oxalate oxidase gene driven by green tissue‐specific promoter increases tolerance to sheath blight pathogen (Rhizoctonia solani) in transgenic rice

Rice sheath blight, caused by the necrotrophic fungus Rhizoctonia solani, is one of the most devastating and intractable diseases of rice, leading to a significant reduction in rice productivity worldwide. In this article, in order to examine sheath blight resistance, we report the generation of transgenic rice lines overexpressing the rice oxalate oxidase 4 (Osoxo4) gene in a green tissue‐specific manner which breaks down oxalic acid (OA), the pathogenesis factor secreted by R. solani. Transgenic plants showed higher enzyme activity of oxalate oxidase (OxO) than nontransgenic control plants, which was visualized by histochemical assays and sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Transgenic rice leaves were more tolerant than control rice leaves to exogenous OA. Transgenic plants showed a higher level of expression of other defence‐related genes in response to pathogen infection. More importantly, transgenic plants exhibited significantly enhanced durable resistance to R. solani. The overexpression of Osoxo4 in rice did not show any detrimental phenotypic or agronomic effect. Our findings indicate that rice OxO can be utilized effectively in plant genetic manipulation for sheath blight resistance, and possibly for resistance to other diseases caused by necrotrophic fungi, especially those that secrete OA. This is the first report of the expression of defence genes in rice in a green tissue‐specific manner for sheath blight resistance.     

Effect of Magnesium on the Development of Sheath blight in Rice

This study investigated the effect of magnesium (Mg) on sheath blight, caused by Rhizoctonia solani, development on rice plants from cultivars BR‐IRGA 409 and Labelle grown in nutrient solution containing 0.062, 0.125, 0.25 and 0.50 mm of Mg. Sheath blight progress on inoculated sheaths was evaluated by measuring lesions expansion (mm) at 24, 48, 72 and 96 h after inoculation. Data were used to calculate the area under lesion expansion progress curve (AULEPC). The relationship between the foliar Mg concentration and the Mg rates was quadratic. The Mg concentration on leaf sheaths tissue was highest at the Mg rates of 0.389 and 0.400 mm , respectively, for cultivars BR‐IRGA 409 and Labelle. A linear model best described the relationship between the AULEPC and the Mg rates. The AULEPC decreased by 48.7 and 26.2% for plants of cultivars BR‐IRGA 409 and Labelle, respectively, as the Mg rates in the nutrient solution increased. The results permitted to conclude that high foliar Mg concentration played a pivotal role to decrease sheath blight lesions expansion.     

Oxalic acid-induced resistance to Rhizoctonia solani in rice is associated with induction of phenolics,peroxidase and pathogenesis-related proteins

Abstract

Oxalic acid (1 mM) when applied as a foliar spray to rice plants induced resistance to challenge infection with Rhizoctonia solani, the rice sheath blight pathogen. Maximum reduction in sheath blight incidence was observed when the plants were sprayed with oxalic acid three days before inoculation with the fungus. The biochemical alterations in rice plants treated with oxalic acid was also investigated. When rice plants were treated with oxalic acid, a two-fold increase in phenolic content in leaf sheaths was recorded three days after treatment. Phenylalanine ammonia-lyase and peroxidase activities increased significantly starting from two days after treatment. Peroxidase (PO) isozyme analysis indicated that PO-3 and PO-4 were induced two days after treatment with oxalic acid. Western blot analysis revealed that two chitinases (28 and 35 kDa) and two β-1,3-glucanases (30 and 32 kDa) were strongly induced in rice sheaths four to six days after treatment with oxalic acid. Immunoblot analysis of protein extracts from oxalic acid-treated plants demonstrated the induction of a 23 kDa thaumatin-like protein (TLP) cross-reacting with bean TLP antibody. These results suggest that the enhanced activities of defense enzymes and defense-related compounds in oxalic acid-treated rice plants may contribute to resistance against R. solani.     

Phenotyping of Partial Physiological Resistance to Rice Sheath Blight

Sheath blight, caused by Rhizoctonia solani, is one of the most important rice diseases worldwide especially under irrigated agro‐ecosystems. To date, no rice accession with complete resistance to sheath blight has been reported. However, a number of genotypes with varying levels of resistance have been reported. Twelve genotypes (including mega varieties) viz. Tetep, Jasmine 85, Te‐Qing, Duduruchi, Betichikon, Khatochalani, D‐6766, D‐256, Swarna, Sarju‐52, MTU‐1010 and Samba Mashuri were evaluated for quantitative measurement of partial physiological resistance to sheath blight under controlled conditions using detached tiller method. Three independent experiments, each involving three replications, were conducted. Seven days after inoculation, the following disease variables were measured: number of lesions, lesion length, vertical sheath colonization (VSC) on the tiller, disease severity, relative vertical sheath colonization (RVSC) and survival of the leaf blade. Variation between rice genotypes was observed for all the disease variables. Disease severity and VSC were the two most correlated variables, whereas the number of lesions and mean lesion length were the least correlated variables. The ranking of varieties often differed depending on the disease variable considered. Amongst the genotypes tested, D‐256, Tetep and Jasmin‐85 had the lowest number of lesions and disease severity. Similarly, Tetep and D‐256 showed the lowest levels of RVSC, whilst Jasmine‐85 was found to be intermediate. D‐6766, Samba Mashuri and Betichikon showed the highest levels of disease variables. The fraction of dead leaves ranged from 0.00 to 0.38. No dead leaves were observed in Te‐Qing, Swarna and MTU‐1010. The highest fraction of dead leaves was observed for Betichikon (0.38) followed by Duduruchi and D‐6766 (0.33). Our results suggest that this method in combination with other phenotyping methods could be used to quantify partial resistance to rice sheath blight.     

水稻株形对纹枯病抗性的影响

利用240份源于珍汕97B/明恢63的重组自交系水稻(Oryza sativa L.)群体,连续2年调查纹枯病病级与水稻生育期、株高和叶片长宽等18个株形性状的关系.对株形性状与纹枯病病级进行了偏相关分析.实验结果,只有植株松紧度与病级表型偏相关两年中都达到了显著或极显著水平,倒2叶基角、穗层整齐度等8个性状与病级之间的偏相关只有一年达显著或极显著水平.结合构建的分子标记遗传连锁图谱,对各性状进行QTL定位.在抗纹枯病QTL相近区间仅检测到控制分蘖角、植株松紧度和倒2叶基角的QTLS,未发现其余株形性状QTLs与抗纹枯病QTLs分布在同一染色体上.结果表明,水稻对纹枯病的抗性主要是由本身抗性基因控制,株形对纹枯病抗性表达的影响主要是间接影响,即通过改变田间小气候而影响发病程度.抗纹枯病育种在累加主效抗纹枯病QTLs的同时,也要注重选择不利于纹枯病发展的株形性状.     

Stability of sheath blight resistance in transgenic ASD16 rice lines expressing a rice <Emphasis Type="Italic">chi11</Emphasis> gene encoding chitinase

Development of transgenic plants by introducing defense genes is one of the strategies to engineer disease resistance. Transgenic ASD16 rice plants harbouring rice chitinase chi11 gene, belonging to a PR-3 group of defense gene conferring sheath blight (Rhizoctonia solani Kuhn) resistance, were used in this study. Three T₂ homozygous lines (ASD16-4-1-1, 5-1-1, and 6-1-1) were identified from seven putative (T₀) transgenic lines expressing chi11 using Western blotting analysis. The inheritance of sheath blight resistance in those lines was studied over generations. The stability of chi11 expression up to T₄ generation in all the three homozygous lines was proved by Western blot and the stability of sheath blight resistance in the homozygous lines was proved up to T₄ generation using detached leaf and intact leaf sheath assays. Among the three homozygous lines tested, ASD16-4-1-1 showed consistent results in all the generations and gave a better protection against the sheath blight pathogen than the other two lines.     

Mean residence time of leaf number, area, mass, and nitrogen in canopy photosynthesis

Mean residence time (MRT) of plant nitrogen (N), which is an indicator of the expected length of time N newly taken up is retained before being lost, is an important component in plant nitrogen use. Here we extend the concept MRT to cover such variables as leaf number, leaf area, leaf dry mass, and nitrogen in the canopy. MRT was calculated from leaf duration (i.e., time integral of standing amount) divided by the total production of leaf variables. We determined MRT in a Xanthium canadense stand established with high or low N availability. The MRT of leaf number may imply longevity of leaves in the canopy. We found that the MRT of leaf area and dry mass were shorter than that of leaf number, while the MRT of leaf N was longer. The relatively longer MRT of leaf N was due to N resorption before leaf shedding. The MRT of all variables was longer at low N availability. Leaf productivity is the rate of canopy photosynthesis per unit amount of leaf variables, and multiplication of leaf productivity by MRT gives the leaf photosynthetic efficiency (canopy photosynthesis per unit production of leaf variables). The photosynthetic efficiency of leaf number implies the lifetime carbon gain of a leaf in the canopy. The analysis of plant-level N use efficiency by evaluating the N productivity and MRT is a well-established approach. Extension of these concepts to leaf number, area, mass, and N in the canopy will clarify the underlying logic in the study of leaf life span, leaf area development, and dry mass and N use in canopy photosynthesis.