Phytosiderophore release as a criterion for genotypic evaluation of iron efficiency in oat

Iron (Fe) deficiency in small grains grown on calcareous soils results in reduced yields, is difficult and expensive to treat with fertilizer, and is complicated to overcome by genetic field screening due to heterogeneous soil and environmental conditions. Recently, phytosiderophore release has been linked to ability of species and genotypes to resist Fe‐deficiency chlorosis. We propose a laboratory technique to measure phytosiderophore release by Fe‐deficient oat (Avena sativa L.) genotypes as a selection method for Fe‐deficiency chlorosis resistance in oat. Plants were grown in Fe‐limiting nutrient solution and phytosiderophore release was measured on 11 days. Summations of daily phytosiderophore release by 17 oat genotypes correlate well with Fe‐deficiency chlorosis scores in the field (r = ‐0.70, p = 0.01). The proposed method consistently identified the genotypes most susceptible to Fe deficiency but did not clearly separate the moderately susceptible genotypes. In these latter genotypes, other factors such as active uptake sites, root growth rate, utilization of acquired Fe, or soil interactions may be modifying factors to phytosiderophore in Fe efficiency. Quantification of phytosiderophore provides a useful selection criterion for oat by eliminating the most inefficient types and with refinement, may become a powerful tool for identifying Fe efficiency in grass crops.     

Genetic Differences in Resistance to Iron Deficiency Chlorosis in Peanut

Iron (Fe) deficiency has been a widespread problem in peanut (Arachis hypogaea L.) grown on calcareous soils of northern China and has resulted in significant yield losses. Field observations showed considerable variability in visual chlorosis symptoms among peanut cultivars in the same soil. The objective of this study was to confirm the genetic differences in resistance to Fe-deficiency chlorosis in peanut and to identify feasible indicators for screening Fe-efficient genotypes. Resistance to Fe chlorosis of sixteen peanut cultivars grown on calcareous soil was evaluated in the field and physiological responses to Fe-deficiency stress were studied in nutrient solution. There were significant differences in resistance to Fe-deficiency chlorosis among the sixteen peanut cultivars tested, which was identified with SPAD readings, active Fe concentrations in young leaves in the early growth stages, and the pod yield. For Fe-resistant peanut cultivars, Fe-reduction capacity and quality of releasing hydrogen ions from roots increased under Fe-deficiency stress. Highly correlated relationships were observed between the summation of root Fe reduction and field chlorosis scores for sixteen cultivars (r² = 0.79). It was concluded that Fe-reduction capacity was a better physiological indicator for screening Fe-efficient peanut genotypes of the mechanisms measured.     

INHERITANCE OF RESISTANCE TO IRON DEFICIENCY CHLOROSIS IN CHICKPEA (CICER ARIETINUM L.)

Iron (Fe)-deficiency chlorosis causes considerable yield losses in chickpea (Cicer arietinum L.) when susceptible genotypes are grown in calcareous soils with high pH. The most feasible method for alleviating Fe deficiency is the selection of suitable cultivars resistant to Fe deficiency chlorosis. ICC 6119 (desi type), which is Fe-deficient chlorosis, was crossed with CA 2969 and Sierra (kabuli types), resistant to Fe deficiency chlorosis. Inheritance of resistance to Fe deficiency in chickpea revealed that the resistance was controlled by a single dominant gene in these genotypes crossed. A negative selection for resistance to Fe deficiency chlorosis will be effective after segregating generations.     

Response of five citrus rootstocks to iron deficiency

Citrus established in calcareous soils can be affected by iron (Fe)‐deficiency chlorosis which limits yield and the farmers' income. The degree of deficiency depends on the rootstock, but the resistance to Fe chlorosis still requires further investigation. To study physiological parameters of citrus rootstocks that could be used to evaluate resistance to Fe deficiency, plants of Troyer citrange (Citrus sinensis L. Osb. × Poncitrus trifoliata L. Raf.), Carrizo citrange, Volkamer lemon (Citrus volkameriana Ten. & Pasq.), alemow (Citrus macrophylla Wester), and sour orange (Citrus aurantium L.) were grown in nutrient solutions with 0, 5, 10, 15, or 20 μM Fe. For each rootstock, plant height, root and shoot dry weights, and concentration of Fe in the shoots and roots were measured at the end of the experiment. Chlorophyll (CHL) concentration was estimated throughout the experimental period using a portable CHL meter (SPAD‐502) calibrated for each rootstock. At the end of the experiment, CHL fluorescence parameters were measured in each rootstock with a portable fluorimeter. Maximal and variable fluorescence values indicated that the photochemistry of Troyer was more affected by a low concentration of Fe in the nutrient solution than that of other rootstocks. To compare rootstocks, the absolute CHL concentration was converted into relative yield by employing a scaling divisor based on the maximum value of total CHL in plants without Fe‐deficiency symptoms. Exponential models were developed to determine the minimum Fe concentration in nutrient solution required to maintain leaf CHL at 50% of the maximum CHL concentration (IC50). Models were also developed to assess the period of time the rootstocks were able to grow under Fe‐stress conditions before they reached IC50. Volkamer lemon and sour orange needed the lowest Fe concentration (between 4 and 5 μM Fe) to maintain IC50, and Troyer citrange had the highest Fe requirement (14 μM Fe). Citrus macrophylla and Carrizo citrange required 7 and 9 μM of Fe, respectively. Similarly, Volkamer lemon and sour orange rootstocks withstood more days under total Fe depletion or with a low concentration of Fe (5 μM Fe in nutrient solution) until they reached IC50, compared to the other rootstocks. The approach used led to a classification of the rootstocks into three categories, regarding their internal tolerance to Fe chlorosis: resistance (sour orange and Volkamer lemon), intermediate resistance (C. macrophylla and Carrizo citrange), and reduced resistance (Troyer citrange).     

A greenhouse technique for screening Trifolium seedlings for iron‐deficiency chlorosis 1

Susceptible Trifolium plants often exhibit symptoms of iron (Fe)‐deficiency chlorosis when grown on high pH, calcareous soils. A greenhouse method was developed to screen seedlings for Fe‐deficiency chlorosis. ‘Yuchi’ arrowleaf (T. vesiculosum Savi.) and ‘Dixie’ crimson (I. incarnatum L.) clover seedlings were grown in “Super Cell”; Cone‐tainers in six calcareous Texas soils differing in Fe and selected other chemical characteristics. At the fourth trifoliolate leaf stage, chlorosis was induced by saturating the soil for a minimum of 2 weeks. The soils differed in their capacity to induce chlorosis in both clovers. Yuchi was more susceptible than Dixie, showing a higher percentage of chlorosis in five of the six soils. The results indicate that this screening method would be a useful tool for studying Fe‐deficiency chlorosis in Trifolium spp.     

Predicting the incidence of iron chlorosis in calcareous soils of southern Spain

Abstract

Iron chlorosis is a serious crop production problem in many calcareous soils of Southern Spain. The objective of this study was to determine which indigenous soil properties (i.e., those which are essentially permanent) were related to Fe chlorosis. Experiments, using two chickpea (Cicer ariethinum L.) cultivars and a sunflower (Helianthus annuuus L.) cultivar, were carried out in a growth chamber with 25 calcareous soils representing widespread Xerofluvents, Xerorthents, Xerochrepts, Haploxeralfs, Rodoxeralfs, Chromoxererts, and Pelloxererts of Southern Spain. The average chlorophyll contents for the three cultivars were significantly correlated with several properties of the carbonate and Fe oxide phases, such as calcium carbonate equivalent (r = 0.69***), “active lime”; (r = 0.58**), acid NH₄‐oxalate extractable Fe (r = 0.68***), Tiron‐extractable Fe (r = 0.61**), and DTPA‐extractable Fe (r = 0.55**). The present and other studies indicate that the soil property most consistently related to Fe chlorosis is acid NH₄‐oxalate extractable Fe (Feo). The Feo critical level separating soils with a high probability from those with a low probability of responding to Fe fertilization was 0.63 g/kg soil, a value similar to those found in other studies. This further supports the use of Feo as a key property to predicting the appearance of Fe chlorosis.     

Modified procedures for commercial adaptation of root iron‐reducing capacity as a screening technique

Abstract

Genotypic evaluation is critical to development of soybean [Glycine max (L.) Merr.] cultivars with genetic resistance to Fe‐deficiency chlorosis. Root Fe³⁺ reducing activity is correlated with genotypic resistance to Fe chlorosis measured in field nurseries, and thus may be a reliable method for identifying chlorosis‐resistat genotypes. However, to develop methods useful for large‐scale screening, several modifications of the previously published procedure for measuring root Fe³⁺ reducing activity were investigated. Several hydroponic experiments were conducted to test proposed modifications. It was determined that: (a) different genotypes may be grown together in the same nutrient solution without affecting Fe³⁺ reduction, (b) genotype separation is maximized by growth in CaCO₃ buffered solution (37.5 mg L^?1), (c) a labor‐intensive elongation step can be eliminated, and (d) denotype evaluation can be accomplished without introducing Fe into the hydroponic solutions. These refinements to the procedure should allow its adaptation and use in soybean breeding programs.     

Iron deficiency in wheat grown on the southern plains

Extensive areas of chlorotic winter wheat (Triticum aestivun L.) were observed on calcareous soils in western Oklahoma. Spraying severely chlorotic wheat with ferrous sulfate (FeSO₄) resulted in greening within a week and doubled herbage yield a month later. Intensive grazing of wheat prior to jointing induced no to severe chlorosis in 33 wheat cultivars tested over three years. After peak intensity in early April, the chlorosis diminished and was not visible when the wheat was fully headed in May. Overall, we believe increased iron (Fe) deficiency in wheat on the Southern Plains is due to increased use of Fe‐inefficient cultivars and stress induced by grazing. Wheat cultivars less susceptible to Fe deficiency are commercially available.     

Identification of iron reductase isozymes in soybeans 1

Native polyacrylamide gel electrophoresis (PAGE) analysis of iron reductase expression in thirteen soybean (Glycine max L.) cultivare revealed the existence of several electrophoretically distinguishable proteins with iron reducing activity. Iron‐deficiency stress may induce expression of proteins with iron reductase activity having Rf values of 0.18 and 0.23 in two iron‐efficient cultivars, A7 and Anoka T, respectively. Variation of iron reductase isozyme expression among cultivars was present, but did not correlate with the iron reducing potential of the cultivars or with field chlorosis resistance ratings. Observation of isozyme expression over time in iron‐efficient A7 and iron‐inefficient T203 revealed expression in A7 of two iron reducing bands absent in T203. However, staining intensity and time of initiation indicate that these bands are not associated with the enhanced iron reduction induced by iron deficiency. Iron reductase isozyme expression cannot account for enhanced iron reduction in A7. It is unlikely that selection for iron reductase isozymes can be used to advance genetic resistance to iron‐deficiency chlorosis in soybeans.     

Soybean genotype evaluation for iron deficiency chlorosis using sodium bicarbonate and tissue culture

The evaluation of soybean genotypes for resistance to iron deficiency through field experiments is complicated by variation in symptom expression. The objective of this study was to develop a tissue culture technique that could distinguish between Fe‐efficient and Fe‐inefficient genotypes. Ten soybean genotypes varying in sensitivity to iron deficiency were planted at 5 locations and rated based on chlorosis expression. For the lab evaluation, friable callus of the ten genotypes was placed on a 4MSII medium amended with 10 mM NaHCO₃. Callus growth reduction relative to a control medium without NaHCO₃ was recorded. Callus weight of all cultivars was affected by the addition of NaHCO₃ however, significant differences in growth reduction among genotypes was observed. The high correlation between callus growth reduction and field chlorosis ratings observed (r²=0.92) and the fact that the expiant source plant can be grown to produce seed indicates that this technique would be useful in a soybean breeding program.     

Factors in iron‐stress response mechanism enhanced by Zn‐deficiency stress in Sanilac,but not Saginaw navy bean

Zinc‐inefficient Sanilac and Zn‐efficient Saginaw navy bean (Phaseolus vulgaris L.) differ in their susceptibility to Zn‐deficiency stress. Sanilac accumulates Fe under Zn‐deficiency stress and Saginaw does not. These two navy bean cultivars were grown at 0, 0.006 and 0.12 mg/L Zn in modified Hoagland nutrient solution. Various Fe‐stress response mechanisms were quantified periodically over a 12‐day experimental period to determine if known factors in the Fe‐stress response mechanism were enhanced by Zn‐deficiency stress. Visual Zn‐deficiency symptoms were more severe in Sanilac than Saginaw navy bean under equivalent Zn treatments. Sanilac contained lower leaf Zn than Saginaw when Zn was present in solution (0.006 and 0.12 mg/L Zn), but the two cultivars were similar in leaf Zn in the absence of Zn (0 mg/L Zn). Sanilac accumulated more leaf Fe than Saginaw when under Zn stress (0 and 0.006 mg/L Zn). The higher levels of leaf Fe in Sanilac than Saginaw were closely associated with enhanced release of reductants and increased reduction of Fe³⁺ to Fe²⁺ by roots of Sanilac. Saginaw navy bean roots reduced Fe³⁺ to Fe²⁺ similarly to Sanilac with adequate Zn present in solution (0.12 mg/L), but experienced minuscule levels of Fe³⁺ reduction under Zn deficiency. Zinc deficiency stimulated the initiation of the Fe‐stress response mechanism in Sanilac, but not Saginaw, which may have enhanced the development of Zn‐deficiency symptoms in Sanilac due to the increased uptake of Fe by this cultivar. The common Fe‐deficiency stress response associated primarily with grasses (release of phytosiderophore) was not found in either navy bean cultivar.     

Factors related to iron uptake by dicotyledonous and monocotyledonous plants. I. pH and reductant

Some plants respond to Fe‐deficiency stress by inducing Fe‐solubilizing reactions at or near the root surface. In their ability to solubilize Fe, dicotyledonous plants are more effective than monocotyledonous plants. In this study we determined how representative plants differ in their response when subjected to Fe‐deficiency stress in a calcareous soil and in nutrient solutions. Iron‐inefficient genotypes of tomato, soybean, oats, and corn all developed Fe chlorosis when grown in soil, whereas Fe‐efficient genotypes of these same species remained green. The same genotypes were grown in complete nutrient solutions and then transferred to nutrient solutions containing N (as NO₃ ^‐) and no Fe.

The T3238 FER tomato (Lycopersican esculentum Mill.) Fe‐efficient) was the only genotype that released significant amounts of H from the roots (the pH was lowered to 3.9) and concomitantly released reductants. Under similar conditions, Hawkeye soyhean [Glycine max (L.) Merr.] released reductants but the solution pH was not lowered. Both Fe‐inefficient and Fe‐efficient genotypes of oats (Avena sativa L.) and corn (Zea mays L.) released insufficient H or reductant from their roots to solubilize Fe; as a result, each of these genotypes developed Fe‐deficiency (chlorosis).

The marked differences observed among these genotypes illustrate the genetic variability inherent within many plant species. A given species or genotype may accordingly not be adapted to a particular soil. Conversely, a given species or genotype may be found (or developed) that is precisely suited for a particular soil. In this event, the need for soil amendments may be reduced or eliminated.     

Differences in Response to Iron Deficiency Among Some Lines of Common Bean

Abstract

Five dry bean cultivars (Coco blanc, Striker, ARA14, SVM29‐21, and BAT477) were evaluated for their resistance to iron deficiency on the basis of chlorosis symptoms, plant growth, capacity to acidify the external medium and the root‐associated Fe³⁺‐reduction activity. Plants were grown in nutrient solution supplied or not with iron, 45 µM Fe(III)EDTA. For all cultivars, plants subjected to iron starvation exhibited Fe‐chlorosis. These symptoms were more severe and more precocious in BAT477 and Coco blanc than in the others cultivars. An important acidification of the culture medium was observed between the 4th and the 8th days of iron starvation in Striker, SVM29‐21 and, particularly, ARA14 plants. However, all Fe‐sufficient plants increased the nutrient solution pH. This capacity of acidification appeared more clearly when protons extrusion was measured in 10 mM KCl + 1 mM CaCl₂. The above genotypic differences were maintained: ARA14 showed the higher acidification followed by Coco blanc and BAT477. Iron deficiency led also to an increase of the root‐associated Fe(III)‐reductase activity in all lines. However, genotypic differences were observed: Striker shows the highest capacity of iron reduction under Fe deficiency condition.     

Depressed growth of non‐chlorotic vine grown in calcareous soil is an iron deficiency symptom prior to leaf chlorosis

The development of iron deficiency symptoms (growth depression and yellowing of the youngest leaves) and the distribution of iron between roots and leaves were investigated in different vine cultivars (Silvaner, Riparia 1G and SO4) grown in calcareous soils. As a control treatment all cultivars were also grown in an acidic soil. Only the cultivars Silvaner and Riparia 1G showed yellowing of the youngest leaves under calcareous soil conditions at the end of the cultivation period. All cultivars including SO4 showed severe shoot growth depression, by 50 % and higher, before yellowing started or without leaf yellowing in the cultivar SO4. Depression of shoot growth occurred independently from that of root growth. In a further treatment the effect of Fe‐EDDHA spraying onto the shoot growth of the cultivar Silvaner after cultivation in calcareous soil was investigated. Prior to Fe application plants were non‐chlorotic, but showed pronounced shoot growth depression. Spraying led to a significant increase in shoot length, though leaf growth was not increased. Accordingly, depression of shoot growth of non‐chlorotic plants under calcareous soil conditions and with ample supply of nutrients and water has been evidenced to be at least partly an iron deficiency symptom. We suggest that plant growth only partially recovered because of dramatic apoplastic leaf Fe inactivation and/ or a high apoplastic pH which may directly impair growth. Since growth was impaired before the youngest leaves showed chlorosis we assume that meristematic growth is more sensitively affected by Fe deficiency than is chlorophyll synthesis and chloroplast development. In spite of high Fe concentrations in roots and leaves of the vines grown in calcareous soils plants suffered from Fe deficiency. The finding of high Fe concentrations also in young, but growth retarded green leaves is a further indication that iron deficiency chlorosis in calcareous soils is caused by primary leaf Fe inactivation. However, in future, only a rigorous study of the dynamic changes of iron and chlorophyll concentration, leaf growth and apoplastic pH at the cellular level during leaf development and yellowing will provide causal insights between leaf iron inactivation, growth depression, and leaf chlorosis.<?show $6#>     

Yield of iron‐sprayed and non‐sprayed strawberry cultivars grown on high ph calcareous soil

Abstract

Iron (Fe) deficiency chlorosis (FeDC) results in extensive reduction in yield of strawberry (Fragaria x ananassa Duch.) grown on high pH calcareous soils. Three cultivars differing in response to FeDC were grown on a high pH (8.2) calcareous soil (25.4% calcium carbonate equivalent in surface 20 cm) in the field (Choueifat, coastal area of Lebanon) to determine the effects of FeDC on fruit yield of cultivars sprayed with FeEDDHA [ferric ethylene‐diiminobis (2‐hydroxyphenyl) acetate]. The unsprayed plots were used as a control. No significant interaction (P<0.05) between cultivars x FeEDDHA spray treatment, and no significant differences (P<0.05) between one and two FeEDDHA spray(s)/week treatment was noted for visual FeDC, fruit number, and fruit yield. Sprayed cultivars once a week produced higher yields than unsprayed ones; overall increases were 33% (13% for ‘Motto’, 30% for ‘Chandler’, and 56% for ‘Douglas'). Even though only slight FeDC was noted on the ‘Motto’ cultivar receiving no Fe EDDHA spray, fruit yields were increased when sprayed with FeEDDHA. However, significant increases in yield for ‘Chandler’ and ‘Douglas’ cultivars with severe FeDC ratings were rioted when sprayed with FeEDDHA.     

Associating SSR Markers with Soybean Resistance to Iron Deficiency Chlorosis

Abstract

Iron deficiency chlorosis (IDC) causes soybean yield loss to growers when certain varieties are planted on calcareous soils. Planting IDC‐resistant varieties is the best management practice, although they may still exhibit chlorosis under certain environmental conditions. Environmental variation for chlorosis expression impedes progress in improving IDC resistance. Breeders could use molecular marker‐assisted selection (MAS), an environment‐independent tool, to improve soybeans' resistance to IDC. Our objective was to determine the efficiency of simple sequence repeat (SSR) markers in selecting for IDC resistance in soybean. A breeding population was developed using parents differing in IDC resistance and yield potential. The population was advanced to the F₂ and F_2:4 generations. Foliar chlorosis data were recorded for parents and F_2:4 lines in replicated field tests planted on calcareous soils at two Iowa locations in 2000 and 2001. Chlorosis scores between parents and F_2:4 lines varied according to location and year. Genotypic data were obtained on individual F₂ plants, and association between chlorosis scores and allele segregation was tested by single‐factor analysis of variance using 2001 data. Three SSR markers were associated (P ≤ 0.1) with chlorosis scores at each location; however, the identity of the markers associated with chlorosis scores was different at each location. In addition, two SSR markers associated with IDC resistance were examined for their efficacy in improving breeding efficiency. Preliminary data presented herein demonstrate the importance of environment on expression of this soil‐stress factor and the potential of using SSR markers as an environment‐independent selection tool for breeding IDC resistance in soybean.     

Effects of phosphate carriers,iron, and indoleacetic acid on iron nutrition and productivity of peanut on a calcareous soil

Results of a field experiment designed to assess the effects of phosphate carriers, iron (Fe), and indoleacetic acid (IAA) on the Fe nutrition of peanut grown on a calcareous soil showed that single superphosphate (SSP) was more effective than diammonium phosphate (DAP) in improving Fe nutrition and chlorophyll synthesis. Increased phosphorus (P) and Fe contents of chlorotic leaves showing symptoms of Fe deficiency suggested that Fe, despite absorption and uptake, was subjected to inactivation, and that the Fe content per se was not the cause of the observed chlorosis. Better amelioration of chlorosis with the SSP treatment as compared with DAP indicated a role of sulphur (S) in preventing inactivation of Fe, possibly caused by excessive P accumulation. A foliar spray of Fe‐EDDHA corrected the chlorosis, but a ferric citrate foliar treatment did not. This further suggested that the mobility of Fe was impaired in chlorotic plants. An IAA foliar spray only also tended to improve Fe nutrition. Significant increase in peanut productivity was observed following improvement in Fe nutrition both with soil and foliar treatments.     

Importance of iron use‐efficiency of nodules in common bean (Phaseolus vulgaris L.) for iron deficiency chlorosis resistance

Several studies suggest that the Fabaceae‐Rhizobium symbiosis is particularly sensitive to iron (Fe) deficiency with respect to NO₃^–‐dependent plants. The aim of this study, which is part of a screening program for common bean tolerance to Fe deficiency, was to study genotypical differences in Fe requirement and Fe use‐efficiency of common bean cultivars depending on symbiotic nitrogen fixation (SNF). Results show that ARA14 produces more whole plant dry matter and particularly more nodule biomass than Coco blanc. ARA14 is characterized by a high capacity of nitrogen fixation and a better Fe use‐efficiency for the growth and the function of the nodules.     

花生缺铁黄化的敏感时期及耐低铁品种的筛选指标

采用盆栽试验,系统研究了石灰性土壤上16个花生品种在各个生育时期新叶的黄化度、叶绿素值、活性铁含量的差异及其动态变化。结果表明,缺铁胁迫下花生耐低铁和铁敏感品种间叶片的黄化程度存在着显著差异,大多数铁敏感品种在出苗后50～65 d时黄化度最高。供试16个品种顶部新展开叶片的叶绿素值（SPAD值）和活性铁含量在整个生育期的变幅分别为4.5～34.6和8.0～36.3 mg/kg, FW,随生长时间的延长两者均呈高―低―高的动态变化趋势。在生长前期,耐低铁品种新叶的叶绿素值和活性铁含量均显著高于铁敏感品种;开花期是花生对缺铁胁迫最为敏感的时期,此阶段黄化现象最严重、各品种新叶的叶绿素值和活性铁含量最低。相关分析表明,在生长前期叶绿素值与黄化度、活性铁及荚果产量之间均呈极显著的相关关系。新叶叶绿素值可作为花生耐低铁品种筛选的一可靠指标。     

Fe‐deficiency stress tolerance and pH reduction: Some contrasts in the cultivars of sesame and lentil

Fe‐deficiency chlorosis was induced in 4 lentil (Lens esculenta Moench) and 7 sesame (Sesamum indicum L.) cultivars by growing them in full nutrient solution for 20 days and then in the nutrient medium without Fe. In lentil cv. VL‐1, a mild chlorosis appeared but turned green after 14 days of stress. However, this recovery was not paralleled by a decrease in pH of the medium. In sesame, there were differences in the degree of tolerance. The cv. T‐13 did not develop any chlorosis, and the pH was found to steadily decrease to 4.5. The cv. SP‐1181 lowered the pH to only 5.6, and did not recover from the chlorosis. A second pattern was noted in VS‐2, TS‐25, TMV‐3 and TMV‐4; these cultivars reduced the pH to between 4.1 and 4.5; these showed a mild chlorosis which disappeared later.

The results show that the pH reduction is not a requisite for chlorosis recovery, at least in some crop cultivars. There are obviously some other mechanism which makes Fe available to the chlorotic leaves. It is suggested that a retranslocatlon of Fe from the older leaves may take place under the stress condition through some physiological process.