尖孢镰刀菌侵染对马铃薯光合效率和叶绿素荧光参数影响

为研究枯萎病对马铃薯光合特性的影响,在幼苗期接种尖孢镰刀菌Fusarium oxysporum后,统计抗病品种陇薯10号和感病品种新大坪的病情指数,并测定叶绿素含量、光合及荧光参数。结果表明,尖孢镰刀菌侵染30 d后马铃薯表现出枯萎病症状,叶绿素含量显著降低,其中叶绿素a含量降幅最大,抗病品种陇薯10号比对照降低9.64%,感病品种新大坪比对照降低14.24%。尖孢镰刀菌侵染后马铃薯光合效率显著降低,侵染30 d后,抗病品种陇薯10号净光合速率比对照降低39.56%,感病品种新大坪比对照降低47.13%。病株的光响应曲线参数光补偿点、暗呼吸速率和表观量子效率都显著提高;而光饱和点和最大净光合速率都显著低于对照,表明尖孢镰刀菌侵染缩小了马铃薯对光能的利用有效范围。病株CO2响应曲线参数CO2饱和点、最大净光合速率和羧化效率分别显著低于对照;病株CO2补偿点和光呼吸速率反而升高,说明碳同化过程受到尖孢镰刀菌的限制。暗适应下的初始荧光、最小荧光、最大荧光、PSII最大光化学效率、光适应下PSII最大光化学效率及实际光化学效率、光化学猝灭系数、非光化学猝灭系数和光合电子传递速率均显著低于对照,...     

黄野螟性信息素分泌腺形态及超微结构

为深入了解黄野螟Heortia vitessoides性信息素生物合成及释放机制,采用光学显微镜、扫描电镜和透射电镜对黄野螟雌成虫性信息素分泌腺形态和超微结构进行观察。结果表明,黄野螟雌成虫性信息素分泌腺为一个完整的环状上皮结构,位于腹部末端第8腹节和第9腹节之间的节间膜上。黄野螟雌成虫性信息素分泌腺表面分布着3类8种感器,即I～IV型毛形感器、I和II型刺形感器及I和II型锥形感器。分泌腺细胞单层密集排列在角质层内表面,细胞呈柱状或圆形,表皮层分为上表皮和内表皮,细胞中还有发达的肌肉层、脂滴以及线粒体等结构组织。     

Colonisation and histological changes in muskmelon and autumn squash tissues infected by <Emphasis Type="Italic">Acremonium cucurbitacearum</Emphasis> or <Emphasis Type="Italic">Monosporascus cannonballus</Emphasis>

Muskmelon (Cucumis melo cv. Temprano Rochet) and autumn squash (Cucurbita maxima) seedlings were inoculated either with Acremonium cucurbitacearum or Monosporascus cannonballus, two of the soil-borne fungi implicated in ‘melon collapse’. Inoculation was achieved in two different ways: by growing the plants in pots containing infested soil to study the histological changes produced in the infected tissues using light microscopy and by growing seedlings in Petri dishes together with fungal colonies in order to observe the colonisation of the plant tissues using scanning electron microscopy. Both muskmelon and autumn squash roots infected with A. cucurbitacearum showed a suberised layer in the epidermis and the outermost layers of the parenchymatic cortex, but these symptoms developed earlier in the muskmelon plants. Muskmelon plants infected by this fungus also presented hypertrophy and hyperplasia, which led to a progressive separation of the vascular bundles in the lower stems of the affected plants. This response was not observed in autumn squash during the study. On the other hand, few histological changes were observed in tissues infected with M. cannonballus and only a slight increase in the size of cortical intercellular spaces was noted in the lower stems of muskmelon plants, and infected autumn squash tissues remained free of these symptoms throughout the study. The scanning electron microscope observations revealed that both fungi were able to colonise the tissues of the two host plants which were studied. A. cucurbitacearum colonised the epidermis and cortex of both muskmelon and autumn squash. The hyphae grew both inter- and intracellularly, and the density of the colonisation decreased within the endodermis. The same colonisation of host plants was observed as a result of M. cannonballus infection. The xylem vessel lumina of both muskmelon and autumn squash showed hyphae and tylose formation as a result of both fungal infections. However, non-fungal structures were detected in the hypocotyl vascular tissues. The present study demonstrates that both fungi are capable of infecting the tissues of a species which is resistant (autumn squash) and a species which is susceptible (muskmelon) to melon collapse.     

Impact of Foliar Diseases on Photosynthesis, Protein Content and Seed Yield of Alfalfa and Efficacy of Fungicide Application

Foliar pathogens attack alfalfa wherever the crop is grown, but their impact, especially on seed production, is poorly understood. In greenhouse trials, leaf spot injury caused by inoculation with various pathogens reduced the crude protein content of infected alfalfa leaves by 22% compared with a healthy control. There was a negative relationship between disease injury and the photosynthetic efficiency of alfalfa plants, as determined by measuring chlorophyll fluorescence in leaves from inoculated vs. non-inoculated seedlings. In field trials at two sites in Alberta from 2001 to 2003, inoculation with Phoma medicaginis increased disease incidence in four of six trials, Phoma sclerotioides increased incidence in four of five trials, and Leptosphaerulina trifolii and Stemphylium botryosum increased incidence in two of six trials. There was a trend for inoculation treatments to reduce seed yield, despite high levels of background infection by indigenous pathogens. The fungicides benomyl and propiconazole inhibited radial growth of Phoma spp. in vitro and reduced disease incidence in inoculated greenhouse experiments. In field trials, applications of benomyl and propiconazole reduced disease incidence, but did not always increase seed yield.     

Cytology of Cork Layer Formation of Citrus and Limited Growth of Elsinoe fawcettii in Scab Lesions

Ultrastructural aspects of host–parasite interactions were investigated in fruits and leaves of citrus (satsuma mandarin) infected with Elsinoe fawcettii. Fungal infection induced host tissues to form cork layers bordering the necrotic areas below the infected sites. The cork layers were composed of compact host cells with convoluted cell walls and alternating lamellations, indicating ligno–suberized tissues in the wound periderm. No host tissues below the cork layers were invaded by hyphae. Hyphae grew intercellularly and intracellularly, often causing hypertrophy and compartmentalization of infected host cells. Also, host cells adjacent to invading hyphae showed accumulation of electron-dense materials and the formation of host cell wall protuberances in intercellular spaces. Hyphae had concentric bodies that showed an electron-transparent core surrounded by an electron-dense layer with radiating filamentous structures on their surface. One or more intrahyphal hyphae were found in the cytoplasm of intercellular or intracellular hyphae. These results suggest that the ligno–suberized cork layers in the wound periderm of citrus act as a protective barrier, which leads to restricted growth of E. fawcettii in bordered scab lesions. The fungus is thought to form concentric bodies and intrahyphal hyphae as a survival mechanism against the water- and nutrient-deficient environments that occur in the cork layers of necrotic host parts.     

盐、碱胁迫对燕麦幼苗光合作用的影响

以砂培‘定莜6号’燕麦幼苗为材料,研究不同浓度Na Cl和Na HCO3(0,50,100,150,200 mmol·L-1)胁迫对光合色素含量、气体交换、叶绿素荧光参数和叶黄素循环的影响。结果表明:(1)在Na Cl胁迫下,随着处理浓度增加,燕麦叶片叶绿素含量、净光合速率(Pn)、气孔导度(Gs)、蒸腾速率(Tr)、PSⅡ最大光化学效率(Fv/Fm)、实际光化学效率(ΦPSⅡ)、光化学猝灭(q P)和Hill反应活力明显下降,非光化学猝灭(NPQ)、调节性能量耗散Y(NPQ)、非调节性能量耗散Y(NO)、激发能分配不平衡偏离系数(β/α-1)、叶黄素循环脱环氧化状态(A+Z)/(V+A+Z)显著提高,类胡萝卜素含量和气孔限制值(Ls)先升后降,胞间CO2浓度(Ci)先降后升。(2)在Na HCO3胁迫下,随着处理浓度增加,燕麦叶片叶绿素含量、Pn、Gs、Tr、Ls、Fv/Fm、ΦPSⅡ、q P和Hill反应活力显著下降,Ci、NPQ、Y(NPQ)、Y(NQ)、(β/α-1)、(A+Z)/(V+A+Z)明显增大,类胡萝卜素先升后降。(3)在相同Na+浓度下,Na HCO3胁迫的燕麦幼苗叶片叶绿素含量、Pn、Gs、Tr、Fv/Fm、ΦPSⅡ、q P和Hill反应活力下降及NPQ、Y(NPQ)、Y(NO)、(β/α-1)、(A+Z)/(V+A+Z)增加的幅度大于Na Cl胁迫。表明盐、碱胁迫均对燕麦幼苗光合系统造成了伤害,但碱胁迫的伤害程度大于盐胁迫。     

黄芩苷对稻瘟病菌细胞生长发育的影响及其对稻瘟病的防效

为研制新型绿色高效稻瘟病防控药剂,分别测定黄芩苷对稻瘟病菌Magnaporthe oryzae附着胞形成、菌落生长、菌丝侵染和致病力的影响以及对稻瘟病的防效。结果显示,黄芩苷抑制稻瘟病菌附着胞形成和菌落生长的最低浓度分别为100 μmol/L和500 μmol/L,在100 μmol/L浓度下黄芩苷能抑制稻瘟病菌侵染菌丝在水稻叶鞘细胞中的扩展及叶片病斑的形成;在盆栽接种试验中,对照组的水稻苗表现为感病和中抗,所占比例分别为38%和62%,而黄芩苷处理组的水稻苗表现为抗病和中抗,其中表现为抗病的水稻苗比例超过70%;黄芩苷处理后,稻瘟病菌分生孢子细胞死亡率比对照组极显著降低。表明黄芩苷通过抑制稻瘟病菌分生孢子的细胞程序性死亡干扰了稻瘟病菌附着胞的形成,从而能有效防控稻瘟病,具有开发为新型绿色高效稻瘟病防控药剂的潜力。     

<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylsphingosine,an inhibitor of sphingosine kinase,induces phytoalexin production and hypersensitive cell death of Solanaceae plants without generation of reactive oxygen species

Plant recognition of elicitors derived from pathogens induces various resistant reactions, including production of reactive oxygen species, hypersensitive cell death and accumulation of phytoalexins. Previously, we isolated a ceramide elicitor from Phytophthora infestans, which activates O₂ ⁻ production of potato suspension-cultured cells. In this study, we employed nine ceramide-related chemicals to test their elicitor activity. Although, none of the tested chemicals induced O₂ ⁻ production, N,N-dimethylsphingosine (DMS) induced accumulation of phytoalexin in potato tubers. In potato, tobacco and Nicotiana benthamiana, DMS also induced rapid cell death. DMS-treated potato cells stained with 4′,6-diamidino-2-phenylindole (DAPI) showed chromatin condensation, and isolated DNA from DMS-treated cells had ladder pattern, confirming that DMS-induced plant cell death is a hypersensitive reaction-like programmed cell death. Involvement of ceramide signaling in induction of plant defense reactions is discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of Essential Oils from Citrus Varieties on in vitro Growth and Sporulation of Phaeoramularia angolensis Causing Citrus Leaf and Fruit Spot Disease

Citrus leaf and fruit spot disease caused by Phaeoramularia angolensis is a serious production constraint in tropical Africa. In previous studies, essential oils extracted from fruit peels of two tolerant varieties exhibited a strong antifungal activity in vitro against P. angolensis as compared to oils from susceptible ones. In order to investigate if the susceptibility of citrus varieties is associated with the antifungal activity of their essential oils, some 22 varieties of different susceptibility levels (tolerant, susceptible and highly susceptible) and belonging to different botanical groups were studied. Oils extracted from fruit peels were evaluated for their activity against radial growth and sporulation using the poisoned food technique. The optimal doses for growth inhibition and conidial reduction were 2500 and 1000 ppm, respectively. At these doses, radial growth and sporulation exceeded the untreated control respectively for four and nine varieties suggesting that oils from these varieties promote fungal development. In general, oils from the tolerant group were most effective in reducing radial growth irrespective of dose. The highly susceptible group ranked first in reducing sporulation at dose 1000 ppm (45.93%) while at higher doses of about 2000–2500 ppm, oils from the tolerant varieties could reduce sporulation up to 100%. The marked dose effect in reducing sporulation suggests that there may be different compounds acting with changing dose. Botanically, oils from pummelo (Citrus maxima, tolerant group), were best in reducing radial growth (>87% inhibition) while those from grapefruits (C. paradisi, highly susceptible group) were most effective in reducing sporulation (>64% reduction).     

The oligopeptide elicitor Pep-13 induces salicylic acid-dependent and -independent defense reactions in potato

The Phytophthora-derived oligopeptide elicitor, Pep-13, originally identified as an inducer of plant defense in the nonhost–pathogen interaction of parsley and Phytophthora sojae, triggers defense responses in potato. In cultured potato cells, Pep-13 treatment results in an oxidative burst and activation of defense genes. Infiltration of Pep-13 into leaves of potato plants induces the accumulation of hydrogen peroxide, defense gene expression and the accumulation of jasmonic and salicylic acids. Derivatives of Pep-13 show similar elicitor activity in parsley and potato, suggesting a receptor-mediated induction of defense response in potato similar to that observed in parsley. However, unlike in parsley, infiltration of Pep-13 into leaves leads to the development of hypersensitive response-like cell death in potato. Interestingly, Pep-13-induced necrosis formation, hydrogen peroxide formation and accumulation of jasmonic acid, but not activation of a subset of defense genes, is dependent on salicylic acid, as shown by infiltration of Pep-13 into leaves of potato plants unable to accumulate salicylic acid. Thus, in a host plant of Phytophthora infestans, Pep-13 is able to elicit salicylic acid-dependent and -independent defense responses.     

不同土壤含水率对红豆杉幼苗叶片气体交换和叶绿素荧光特性的影响

以太行红豆杉幼苗为材料,将土壤含水率阈值分别设置为田间持水率的90%～100%(W1)、80%～90%(W2)、70%～80%(W3)、60%～70%(W4)和50%～60%(W5),定时测量红豆杉幼苗的叶片气体交换参数和叶绿素荧光参数,并于试验后期测量红豆杉幼苗的生长状况。结果表明:红豆杉幼苗叶片叶绿素相对含量、气孔导度、净光合速率和蒸腾速率在W2处理达到最大值,分别比W5增长了10.17%、35.91%、53.03%和85.60%。且W2土壤含水率处理下红豆杉幼苗叶片qP、ETR和Y(Ⅱ)值最高,分别比W5增长了15.38%、30.74%和21.76%。随着土壤含水率的下降,红豆杉幼苗叶片NPQ升高,叶片热耗散能力增强,W5的NPQ值达1.4347,比W1增长了36.78%。W2显著提升了叶片rETR_max和半饱和光强I_k,与W1、W3、W4和W5相比,rETR_max分别增长了24.40%、4.93%、12.60%和26.15%;I_k分别增长了25.06%、7.70%、18.20%和27.08%...     

Purification and partial characterization of xylanase from the fungal maize pathogenHelminthosporium turcicum (Pass)

The fungal pathogenHelminthosporium turcicum was found to secret xylanase when grown on minimal medium containing xylans, wheat straw or isolated maize cell walls. The highest xylanase activity occurre when the fungus was grown on maize cell walls. When glucose was added to this medium xylanase activity was suppressed. The xylanase enzyme was purified from the culture filtrate by subsequent anion exchange chromatography, cation exchange chromatography and gel filtration. The purified xylanase gave a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis corresponding to an apparent molecular weight of 22.5 kDa. It is determined to have a pI of 7.4, specific activity of 11300 nanokatals mg^–1, pH optimum between pH 5.5 and 6.5 and optimal temperature between 50 °C and 60 °C. The half-life of the enzyme at pH 6.0 and 50 °C was found to be 35 min. For primary structure comparison with other xylanases, the protein was digested with trypsin and the resulting peptides were separated by reversed phase chromatography and selected peptides were sequenced. The determined amino acid sequence showed high homology with xylanase fromCochliobolus carbonum and three other fungal xylanases.     

微生物挥发性有机化合物对指状青霉产孢的抑制及其机制

为有效防控柑橘绿霉病,分别测定丙酸乙酯、异丁酸乙酯、2-甲基丁酸甲酯、异丁酸和2-甲基-1-丁醇5种微生物挥发性有机化合物(volatile organic compound,VOC)单剂及其混剂(5种VOC体积比为1:1:1:1:1)对指状青霉Penicillium digitatum菌落生长和产孢的抑制作用、对指状青霉产孢关键调控基因表达量的影响和对分生孢子梗形态的影响及对柑橘绿霉病的室内防控效果。结果显示,丙酸乙酯、2-甲基-1-丁醇单剂和混剂对指状青霉菌丝生长有明显的抑制作用,EC₅₀分别为1.816、0.154和0.230μL/cm³;5种VOC单剂及五者混剂对指状青霉产孢均有不同程度的抑制作用,其中混剂对指状青霉产孢的抑制作用最明显,EC₅₀达到0.006μL/cm³;5种VOC单剂处理后,指状青霉帚状枝变短,侧枝发育不良;一定浓度丙酸乙酯、异丁酸乙酯、2-甲基丁酸甲酯或异丁酸处理后,指状青霉体内brlA和abaA基因过表达,然而2-甲基-1-丁醇和混剂处理后,其体内brlA、abaA和...     

Broad bean mottle virus: Identification,host range,serology, and occurrence on faba bean (Vicia faba) in West Asia and North Africa

One of the faba bean viruses found in West Asia and North Africa was identified as broad bean mottle virus (BBMV) by host reactions, particle morphology and size, serology, and granular, often vesiculated cytoplasmic inclusions. Detailed research on four isolates, one each from Morocco, Tunisia, Sudan and Syria, provided new information on the virus.The isolates, though indistinguishable in ELISA or gel-diffusion tests, differed slightly in host range and symptoms. Twenty-one species (12 legumes and 9 non-legumes) out of 27 tested were systemically infected, and 14 of these by all four isolates. Infection in several species was symptomless, but major legumes such as chickpea, lentil and especially pea, suffered severely from infection. All 23 genotypes of faba bean, 2 of chickpea, 4 of lentil, 11 out of 21 ofPhaseolus bean, and 16 out of 17 of pea were systemically sensitive to the virus. Twelve plant species were found to be new potential hosts and cucumber a new local-lesion test plant of the virus.BBMV particles occurred in faba bean plants in very high concentrations and seed transmission in this species (1.37%) was confirmed.An isolate from Syria was purified and two antisera were produced, one of which was used in ELISA to detect BBMV in faba bean field samples. Two hundred and three out of the 789 samples with symptoms suggestive of virus infection collected in 1985, 1986 and 1987, were found infected with BBMV: 4 out of 70 (4/70) tested samples from Egypt, 0/44 from Lebanon, 1/15 from Morocco, 46/254 from Sudan, 72/269 from Syria and 80/137 from Tunisia. This is the first report on its occurrence in Egypt, Syria and Tunisia. The virus is a potential threat to crop improvement in the region.Samenvatting Eén van de in West-Azië en Noord-Afrika in faba-boon aangetroffen virussen werd geïdentificeerd als het tuinbonevlekkenvirus (broad bean mottle virus) op grond van waardplantreacties, deeltjesvorm en-grootte, serologische eigenschappen en granulaire, vaak gevacuoliseerde celinsluitsels. Verder onderzoek aan vier isolaten uit respectievelijk Marokko, Tunesië, Soedan en Syrië verschafte nieuwe informatie, over het virus.De in ELISA of gel-diffusietoetsen serologisch niet te onderscheiden isolaten verschilden enigszins in waardplantenreeks en symptomen. Van 27 getoetste plantesoorten werden 21 systemisch geïnfecteerd (12 vlinderbloemigen, en 9 niet-vlinderbloemigen) waarvan 14 door alle vier isolaten. In vele ervan was de infectie symptoomloos, maar belangrijke als gewas geteelde vlinderbloemigen, zoals erwt, linzen en kekererwt, leden ernstig onder aantasting. Alle 23 getoetste faba-boongenotypen, beide van kekererwt, alle vier van linzen, 11 van de 21 getoetste vanPhaseolus-boon en 16 van de 17 van erwt bleken systemisch gevoelig voor het virus. Twaalf plantesoorten, bleken nieuwe potentiële waardplanten en komkommer een nieuwe lokale-lesietoetsplant voor het virus te zijn.In faba-boneplanten kwam, het virus in hoge concentratie voor en overdracht met zaad (1.37%) in deze soort kon worden bevestigd.Een Syrisch isolaat werd gezuiverd en twee antisera werden bereid, waarvan één werd gebruikt voor de detectie van het virus in te velde verzamelde monsters. Van 789 in 1985 tot en met 1987 verzamelde bladmonsters, met symptomen die deden denken aan virusinfectie, bleken 203 het virus te bevatten en wel 4 van de 70 (4/70) uit Egypte, 0/44 uit Libanon, 1/15 uit Marokko, 46/254 uit Soedan, 72/269 uit Syrië en 80/137 uit Tunesië. Het virus was nog niet eerder aangetoond in Egypte, Syrië en Tunesië.De grote verbreiding, grote kunstmatige waardplantenreeks, overdracht met zaad, en pathogeniteit voor een aantal belangrijke vlinderbloemige gewassen maken het virus tot een potentiële bedreiging van de programma's tot verbetering van de teelt van de bedoelde gewassen in het betrokken gebied.     

Quantifying and Modelling the Mobilisation of Inoculum from Diseased Leaves and Infected Defoliated Tissues in Epidemics of Angular Leaf Spot of Bean

Daily multiplication factor (number of daughter lesions per mother lesion per day) values were experimentally measured in four replications of a monocyclic experiment on angular leaf spot (ALS) of bean, where sources of inoculum were artificially established within a bean canopy, on the ground (defoliated infected leaves), or both. Daily multiplication factor of lesions in the canopy (DMFRc) was higher than that of infectious, defoliated tissues (DMFRd) in all replications. Both DMFRc and DMFRd were strongly reduced under dry compared to rainy conditions. Under rainy conditions for spore dispersal DMFRd was about two to three times smaller than DMFRc. Defoliated leaves may nevertheless represent a significant source of infection, depending on the amount of infectious tissues. Mother lesions within the canopy generated more daughter lesions in the medium (or lower) layers of the canopy than at its upper level (DMFRc higher at the medium and lower layers of a canopy), whereas DMFRd values seemed to decrease with height in the canopy. A mechanistic simulation model that combines host growth and disease-induced defoliation was designed to simulate the respective contributions of the two components of the dual inoculum source of a diseased canopy (infected foliage and defoliated infectious tissues), and varying infectious periods in both sources. Simulations suggest that higher DMFRc values have a large polycyclic effect on epidemics whereas that of DMFRd is small, and that large effects of the infectious period of lesions in the canopy are found when DMFRc is high. Simulations using experimentally measured DMFRc and DMFRd values indicated much stronger epidemics in rainy compared to dry conditions for spore dispersal, but disease persistence in the latter. The implications of considering a dual source of inoculum in the course of a polycyclic process are discussed with respect to epidemic thresholds.     

Phytoplasma [Stolbur-subgroup (Bois Noir-BN)] infection inhibits photosynthetic pigments, ribulose-1,5-bisphosphate carboxylase and photosynthetic activities in field grown grapevine (Vitis vinifera L. cv. Chardonnay) leaves

In this work we have studied the influence of phytoplasma-induced grapevine yellows (yellowing) on some features of the thylakoids from field grown grapevine (Vitis vinifera L. cv. Chardonnay) leaves. Changes in photosynthetic pigments, soluble proteins, ribulose-1,5-bisphosphate carboxylase, photosynthetic activities and thylakoid membrane proteins were investigated. The level of total chlorophyll and carotenoids, on a unit fresh weight basis, showed a progressive decrease in phytoplasma infected leaves. Similar results were also observed for total soluble proteins and ribulose-1,5-bisphosphate carboxylase activity. When various photosynthetic activities were followed in isolated thylakoids, phytoplasma infection caused marked inhibition of whole chain and photosystem (PS) II activity. Smaller inhibition of PSI activity was observed even in severely infected leaves. The artificial exogenous electron donors, DPC and NH₂OH significantly restored the PSII activity in both mild and severely infected leaves. The same results were obtained when F_v/ F_m was evaluated by Chl fluorescence measurements. The marked loss of PS II activity in infected leaves was evidently due to the loss of 33, 28–25, 23, 17 and 10 kDa polypeptides. This conclusion was confirmed by immunological studies showing that the content of the 33 kDa protein of the water-splitting complex was diminished significantly in infected leaves. Phytoplasma infection induced a fast degradation of LHCP II which became visible as yellowish colour in leaves.