LOTUS NEWSLETTER

1997 Volume 28


CONTENTS

1. Introduction: instructions to contributors notes from the editor

2. Lotus Activities: reports and abstracts

3. Recent Current Literature


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LOTUS NEWSLETTER, No. 28 1997

Purpose: The Lotus Newsletter consists of informal communications of research information on Lotus spp. Reports of any phase of research on Lotus breeding, genetics, taxonomy, management, utilization or physiology are welcome. Your biographic sketches and information about your research objectives, approaches, and progress including titles of your publications are encouraged. Seed requests and news items are accepted.

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Lotus Newsletter

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SPECIAL THANKS

The illustration on the cover is of Lotus glaber Mill. has again been graciously provided by Ana Arambarri (Argentina) . The Lotus illustration is the 5th in a series of illustrations that started with Volume 23. Many scientists are familiar with the name of this species to be Lotus tenuis Waldst. et Kit. Note that according to the International Code of Botanical Nomenclature the correct nomenclature is Lotus glaber Mill.


COMPARATIVE ANALYSIS OF STOMATAL CHARACTERS IN TWO PHENOTYPES WIDELY DIFFERENTIATED IN A POPULATION OF LOTUS GLABER MILL. [SYN. L. TENUIS WALDST. ET KIT (FABACEAE)]

M Colares1 and MM Mujica2

1Area de Botánica
2Area de Genética y Mejoramiento Animal y Vegetal Facultad de Ciencias
Agrarias y Forestales
UNLP, CC 31 (1900) La Plata, Argentina

Introduction

In a plant breeding material of Lotus glaber it was observed, in two successive generations, the presence of a lower frequency of individuals that were differentiated with regard to the typical phenotype by numerous characteristics. Among these are underlined waved branches and larger leaflets. Individuals of both phenotypes were cloned and it has been begun a comparative study of characters with possible agronomic importance.

According to Tichá (1985), the stomatal density and size are important anatomical parameters that contribute to the leaf resistance (conductance) to CO2 and water vapour transport, determining the efficiency in the use of water. The ratio of stomata density in the adaxial/abaxial leaf surfaces also has been registered in various species. This information has been used by Willmer (1985) and Lüttge et al. (1993) for to classify the leaves of the plants as: amphistomatic (present their stomata in both surfaces, but generally they are more numerous in the abaxial surface; as in the majority of herbaceous plants ); hypostomatic (stomata are only on the abaxial leaf surface of arboreal species; epistomatic (stomata are only on the adaxial surface; it is presented by aquatic plants with floating leaves). In typical plants of L. glaber has been determined that the stomata are predominantly on the adaxial surface (Arambarri and Colares, 1993).The objective of this experience was to analyze comparatively the density, dimensions and the distribution adaxial/abaxial of the stomata of two contrasting phenotypes highly differentiated in a L. glaber population.

Materials and methods

It was analyzed a typic phenotype (TP) and a phenotype highly differentiated (DP), found in two successive generations of a population in process of selection. Plants of both types were cloned by stem cutting and were cultivated in pots with homogeneous ground, in natural conditions. A plant of each type was used for this study.

The leaves were obtained of the middle sector of branches of each plant. The epidermis was removed from the third middle of the central leaflet of fresh leaves by the peeling technique. The adaxial or upper epidermis (UE) and abaxial or lower epidermis (LE) were extracted from the same sector but of different leaves. The epidermal samples were mounted in 90% glycerine. The observations of both epidermic samples were fulfilled with a Leitz microscope equipped with a lucid camera. Stomata number was register in 50 samples of each epidermis of different leaves. Stomata width and length were measured in 10 stomata from each sample.

The evaluated stomata characters on both epidermis were: density (number . cm-2), width and length (µm), the ratio length / width and the density ratio UE /LE. Due to the fact that UE and LE not belonged to the same leaf, the analysis of the density ratio UE /LE was on four whole pairs at random combined of the n=50 stomata density data determined on UE and LE.

Data of each character were analyzed statistically and it was applied the "t" test to validate the null hypothesis (H0): DP is not different of TP. Besides were determined the coefficients of single correlation among the characters.

Results and discussion

Table 1 indicates that were found significant differences (t test, P < 0,01) among DP and TP for the characters: stomata density on UE and LE, length of the stomata in UE and the ratio width /length of the stomata in UE.

The stomata density and the stomata size found in both epidermal surfaces of TP was very approaching to those that had been communicated by Arambarri and Colares (1993). The DP showed lower stomata density on both epidermis and stomata on UE longer than the TP plant. This "dilution" of stomata density of DP has been apparently balanced by the increasing size, in length of individual stomata, but it was only on the upper epidermis. In addition to this, similar changes in stomata characters has been associated with a increase in the ploidy level (Willmer, 1983).

The variation coefficient indicated that was a wide variation among the analyzed samples for the stomata density on UE and LE surfaces, especially in the case of DP. By the contrary, the coefficient of variation was low for the width and length of the stomata, especially in the case of DP. These results point out that the stomata size was more constant character than stomata density and that this pattern to become more accentuated in DP than TP.

On the other hand, the stomata density ratio UE /LE, the Table 2 shows that the analysis based on random combinations UE-LE density data denote significant differences among DP and TP (test of "t", P < 0,01). In L. glaber has been determined that the leaves present greater stomatal number in the upper epidermis (Arambarri and Colares, 1993). This sort of distribution adaxial/abaxial of stomata was confirmed in this experience and more over it was found that in the "new" (DP) phenotype was significantly more remarkable (Table 2). Stomata distribution on adaxial/abaxial epidermis was related with the loss of water from leaves that is greater from the stomata of adaxial surface (Robins et al , 1965). According this interpretation the DP plants presents a ratio that would be favorable to a greater loss of water than TP plants.

It was determined by the analysis of single correlation that in TP the analyzed characters varied in independent form (P > 0,05), while in DP only was significant the correlation width vs. length of the LE stomata, (r=0,48, P=0,0003).

Results indicate that the phenotype "new" (DP) present differences from the normal (TP) in the stomata density, stomata length on the UE and density ratio UE /LE. The precedents on the origin of the evaluated materials and the uniformity of the environment conditions during their development permit to suppose that the registered differences could be genetics. This will be studied in future experiences.

Acknowledgments

The authors wish to express their appreciation to Prof. Ana María Arambarri, Area de Botánica, for her assistance.

References

Arambarri AM and M Colares, 1993 L. corniculatus L. and L. tenuis Waldst. et Kit (Leguminosae) anatomy of the leaf. Lotus Newsletter, 24: 38-39.

Tichá I, 1985 Ontogeny of leaf morphology and anatomy. In: Photosynthesis during leaf development, De. Zdenek Sestak. 16-50.

Lüttge U., M. Kluge and G. Bauer, 1993. Botánica. McGraw Hill/Interamericana de España, Madrid. 573 pp.

Willmer C M, 1983. Stamata. Longman Group Limited. London. 192 pp.

Robbins WW, TE Weier and CR Stocking, 1965. Transpiration, conduction and absortion. In: Botany. An introduction to plant science. John Wiley & Sons, Inc., third edition. 167-181.

Table 1. Means and variation parameters of the foliar stomata characters in two contrastant phenotypes of Lotus glaber (TP= typical phenotype and DP= differentiated phenotype). The characters were determined on upper epidermis (UE) and on lower epidermis (LE). n=50
Characters
Means
Standard deviation
Range
Variation coefficient
Stomata density on UE (no. cm-2) TP: 20,720.1**
DP: 16,845.1
4,896.7
5,073.97
12,195-31,707
5,284-28,861
23.63
30.12
Stomata density on LE (no. cm-2) TP: 15,698,8**
DP: 8,072.74
3,953.07
3,996.36
8,943-26,829
2,845-19,105
25.18
40.83
Stomata length on UE (µm)TP: 22.6**
DP: 25.72
2.52
1.49
20-25
20.5-29
11.16
5.78
Stomata width on UE (µm)TP: 20.01 n.s.
DP: 20.05
1.23
0,31
15-25
19.5-21
6.13
1.53
Width/length ratio on UETP: 0.89**
DP: 0.77
0.10
0.04
0.75-1
0.69-0.91
11.37
4.94
Stomata length on LETP: 23.9 n.s.
DP: 24.02
2.53
1.38
20-30
21.5-28.5
10.6
5.76
Stomata width on LETP: 20.3 n.s.
DP: 19.7
1.56
0.61
15-25
17-20.5
7.72
3.07
Width/length ratio on LETP: 0.86 n.s
DP: 0.82
0.10
0.04
0.34-1
0.7-0.93
11.97
4.49


(**): indicated significant differences (t test, P < 0,01), between the means of TP and DP for each character.
(n.s.): differences are not significant.

Table 2. Distribution adaxial/abaxial of stomata determined by density ratio (stomata density on upper epidermis/stomata density on lower epidermis).
(A), (B), (C) and (D) are whole of random combinations (n=50) of stomata density on upper epidermis and lower epidermis, obtained of different leaflets. TP and DP are typical and differentiated phenotypes of L. glaber, respectively.
Whole of random combinations
Average
(X)
Standard deviation
Range
Variation
coefficient
(A)TP

(A)DP
1.40**

2.46
0.45

1.41
0.77 - 2.49

0.69 - 6.96
32.08

57.43
(B)TP

(B)DP
1.43**

2.41
0.57

1.50
0.64 - 2.95

0.57 - 10.14
40.00

62.19
(C)TP

(C)DP
1.31**

2.43
0.49

1.47
0.65 - 3.36

0.64 - 7.71
37.88

60.26
(D)TP

(D)DP
1.39**

2.48
0.47

1.43
0.6 - 3.31

0.46 - 8
33.91

57.50


(**): difference between XTP and XDP is significant (t test, P<0.01)


CHANGES IN THE RELATIVE CONCENTRATIONS OF FLAVONOIDS RELEASED NATURALLY FROM LOTUS GLABER (SYN. L. TENUIS) SEEDS DURING GERMINATION

Wagner M.L.1,2, Kade M.3, Strittmatter C.D.3, Ricco R.A.1 and Gurni A.A.1

1Cátedra de Fármacobotánica
2Museo de Farmacobotánica "Juan A. Domínguez"
Fac. de Farmacia and Bioquímica (UBA)
Junín 956
1113 Buenos Aires. Argentina.

3CEVEG-CONICET
Serrano 669
1414 Buenos Aires. Argentina.

Lotus glaber Mill. (syn. L. tenuis Waldst. et Kit ex Willd). is the only important pasture legume for the Flooding Pampa (Depresión del Río Salado), the most extensive region for calf production in Argentina (7.5 million hect.). The species has perfectly adapted to the unfavorable environmental conditions of this area, to such an extent that during spring and summer it is a dominant species in the natural grasslands that cover the region (Collantes et al. 1988).

Our previous studies in birdsfoot trefoil have demonstrated the dependence of flavonoid composition on developmental stage (Strittmatter et al. 1992, 1994; Wagner et al. 1996). Kaempferol and its glycosides were identified during the whole life cycle of the plant in different organs, whereas free quercetin (Q) and its two glycosides could only be detected in the seeds of L. glaber (Strittmatter et al. 1994; Wagner et al. 1996).

In the present study, the identification of seed flavonoid was completed including the isoflavones. Their distribution was examined during germination, as well as in exudates from seeds, in order to evaluate the role of these flavonoids in response to soil bacteria.

Experimental procedures

Lotus glaber cv. Tresur Chajá, the only registered cultivar in Argentina, was used. Five grams of authentic seeds were extracted with boiling water during 30 min. The aqueous solution was subjected to reduced pressure and the residue redissolved in methanol.

Other 5 g from the same seed lot were washed with a mixture of hydrogen peroxide 100 vol. and methanol 1:1 for 7 min. After rinsing in sterile water, seeds were transferred to a refrigerator (4C) in 50 ml of sterile water during 72 h. This water was evaporated and the residue redissolved in methanol.

The imbibed seeds were then transferred onto pre-wetted filter paper in sterile Petri dishes. Seeds were then germinated at 25C under fluorescent light with a 16 h photoperiod during 4 days. The exudates from germinated seeds were collected by extracting the filter papers with 50 ml of distilled water. This aqueous solution was evaporated under reduced pressure and the residue redissolved in methanol. The germinated seeds were submitted to the same extraction procedure as described previously for dry seeds.

The isolation and purification of flavonoids was achieved by descending chromatography using HOAc 15% on 3 MM Whatman paper. Each band was eluted with methanol and chromatographied again in BAW (n butanol-HOAc-H2O, 4:1:5, upper phase). The obtained compounds were eluted in methanol and analysed using standard procedures (Mabry, Markham and Thomas, 1970; Markham, 1982 and Waterman and Mole, 1994).

The experiments were performed twice with similar results.

Results

When dry seeds of L. glaber were tested for their flavonoid composition, not only kaempferol (K), quercetin (Q), quercetin-3-O-galactoside (QGa) and quercetin-3-O-glucoside (QGl) were identified (Wagner et al. 1996); a kaempferol-3-O-triglucosyl-7-O-rhamnoside (KSR) and a possibly polysubstituted isoflavone (I) were also detected. The structure of this isoflavone will be confirmed with MS techniques. These latter metabolites (KSR and I) are extremely soluble in water as shown by their high Rf values (0.77 and 0.95, respectively) (Table 1).

Table 1: Rf values of the different compounds
Compounds
Rf in water
quercetin-3-O-glucoside (QGl)
.09
quercetin-3-O-galactoside (QGa)
.10
kaempferol-3-O-sophoroside-7-O-rhamnoside (KSR)
.77
isoflavone (I)
.95

The results indicate that when relative concentrations of each flavonoid were compared (Table 2), dry seeds (control) and the filter papers showed the highest concentration of all compounds analysed. Aglycones were released during germination, wheras it was possible to detect KSR and I in the water used for the imbibition of the seeds, because of their high solubility.

After a period of 4 days all flavonoids remained in the germinated seeds in their lowest relative concentration (Table 2).

Table 2: Relative concentrations of flavonoids in Lotus glaber seeds during the first stages of germination
Compounds
Dry seeds (control)
Seed exudates (water)
Germinated seeds exudates (filter paper)
Germinated seeds
K
+
(+)
+
+
Q
++
+
++
+
QGa
++
+
++
+
QG
+++
++
+++
+
KSR
++
++
++
+
I
++
++
++
+
(+ ) traces; + presence; ++ abundance; +++ high abundance

Discussion

Analysis of the flavonoids released during imbibition and germination of L. glaber seeds confirmed our previous results (Wagner et al. 1996) that Q and its glycosides are the dominant flavonoids in the seeds.

These results agree with Hartwig et al. (1991) and Tsai and Phillips (1991), who detected that nearly half (46%) of the flavonoids fraction released from intact alfalfa seeds consisted of Q or its derivatives.

The presence of an isoflavone in dry seeds of L. glaber is also noteworthy. Bonde et al. (1973) and Ingham (1977) reported the isolation of the two isoflavans sativan and vestitol in leaves of L. corniculatus, a closely related species to L. glaber, but in response to inoculations with fungi. Flavonols and isoflavones released from L. glaber seeds were also identified in dry seeds, and thus, they would not function as phytoalexins; they must have been synthesized previously.

It remains to be determined which seed tissue is the source of the mentioned flavonoids; alfalfa seeds most apparently store the flavonoids in the seed coat (Hartwig and Phillips, 1991), whereas in soybean the amounts of isoflavones were much larger in the cotyledons than in the roots or hypocotyls (Suganuma and Takaki, 1992).

Data from this study suggest the following preliminary model of how flavonoids diffuse from seeds during the initial stages of germination. Seeds release the isoflavone and KSR during imbibition into aqueous solution as well as Q glycosides. The aglycones, which are less soluble in water than glycosides, diffuse away from the already germinating seed as revealed the filter paper (Table 2).

Although it could be questionable to extrapolate directly the results from this experiment to the soil environment, the mentioned flavonoids should certainly affect soil microorganisms.

References
Bonde, M.R.; Millar, R.L. and Inham, J.L. 1973. Phytochemistry 12: 2957-2959.

Collantes, M.B.; Kade, M.; Miaczynski, C. and Santanatoglia, O. 1988. Studia Oecologica V: 77-93.

Hartwig, V.A.; Joseph, C.M. and Phillips, D.A. 1991. Plant Physiol. 95: 797-803.

Ingham, J.L. 1977. Phytochemistry 16: 1279-1282.

Mabry, T.J., K.R. Markham and M.B. Thomas. 1970.The Systematic Identification of the Flavonoids, Springer-Verlag, Berlin, New York. pp. 1-175.

Markham, K.R. 1982."Techniques of Flavonoid Identification, Academic Press ed. pp. 1-113.

Strittmatter C.D., Wagner M.L., Kade M. and Gurni A.A. 1992. Bioch. Syst. and Ecol. 20 (7): 685-687.

Strittmatter C.D., Wagner M.L., Kade M., Rivero M., Ricco R.A. and Gurni A.A .1994. Proceedings of The First International Lotus Symposium pp. 199-202.

Suganuma, N. and Takaki,M. 1993. Soil Sci. Plant Nutr. 39 (4): 661-667.

Tsai, S.M. and Phillips, D.A. 1991. Appl. Environ. Microbiol. 57 (5): 1485-1488.

Wagner M.L., Strittmatter C.D., Kade M., Rivero M., Ricco R.A. and Gurni A.A. 1996. YTON 58(1/2): 141-146.

Waterman, P.G. and S. Mole. 1994. Analysis of Phenolic Plant Metabolites, Blackwell Scientific Publications pp. 1-238.


EFFECTS OF FLOODING ON THE REPRODUCTION OF LOTUS GLABER (SYN. L. TENUIS) AND LOTUS CORNICULATUS

Osvaldo R. Vignolio, Néstor O. Maceira and Osvaldo N. Fernández

Cátedra de Ecología
Unidad Integrada Balcarce FCA­UNMdP/EEA-INTA
C.C. 276, 7620 Balcarce, Argentina.

Flooding effects on the reproduction of Lotus glaber Mill. and Lotus corniculatus L. were studied under experimental conditions. The hypothesis that, the effects of flooding on the reproduction differ with species and plant age, was tested.

Flooding were carried out in winter (42 days flooding), spring (49 days) and summer (17 days),in pots under field conditions. Spring flooding was applied to young (133 days) and old (208 days) plants. Winter flooding did not affect weight and number of fruits and seeds per plant. Reproductive characteristics in plants of different age and treatment of the same species were not affected by spring flooding. Lotus glaber produced more fruits and seeds than Lotus corniculatus in the spring flooding.

Indeterminate reproduction and higher reproductive effort of young plants, determined that reproductive traits for each species had no significant differences between treatments and plant age. Summer flooding (17 days) promoted both flower and fruit abortion and a plant mortality of 100 % in L. corniculatus and 50 % in L. glaber.

EFFECTS OF FLOODING ON THE GERMINATION OF LOTUS GLABER (SYN. L. TENUIS) AND LOTUS CORNICULATUS SEEDS. THEIR ROLE IN THE DIFFERENTIAL DISTRIBUTION OF BOTH SPECIES IN THE FLOODING PAMPA (BUENOS AIRES, ARGENTINA).

Lotus glaber (L.g.) and Lotus corniculatus (L.c.) were introduced as forage plants in the Flooding Pampa around 50 years ago. While L.g. has naturally colonized the frequently flooded habitats. L.c. has not shown the same expansion. In this paper we studied the tolerance to flooding of seeds of both species. The seeds were flooded during 7 weeks, in controlled conditions of temperature and light, and samples were taken at increasing time intervals. Germination percentage (G.P.) of the seeds was taken as a measure of tolerance to flooding and it was found that although G.P. decreased with flooding time in both species, the soft seeds of L.c. lost viability earlier than those of L.g. With 49 days of flooding, hard seeds were the only ones that maintained the G.P. and were more frequent in L.g. (approximately 50 % in L.g. and 6 % in L.c., in 5 months old seed). These characteristics probably play an important role in the capacity of L.g. of colonizing habitats that are periodically flooded.

Ecología Austral Vol.5, No 2: 157-163, 1995.


STUDIES ON THE SYMBIOTIC POTENTIAL OF A RHIZOBIUM LOTI STRAIN NATIVE FROM CHASCOMÚS ON LOTUS PEDUNCULATUS AND LOTUS CORNICULATUS VAR. HIRSUTUS PLANTS

M. Julia Estrella, Fernando L. Pieckenstain, Rodolfo A. Ugalde and Alberto A. Iglesias.

Instituto Tecnológico de Chascomús (INTECH - CONICET)
Camino Circunv. Laguna
km 6. Casilla de Correo 164
Chascomús (7130). Bs. As. Argentina.

INTRODUCTION

The Salado River Basin is an extense area in Buenos Aires province (Argentina) dedicated to beef and dairy cattle production, which is mainly sustained on a grazing system based on native grasslands with a minor portion of cultivated pastures. Therefore, proven the extensive conditions of cattle production in the area, nitrogen contents of the forage biomass is a critical point determining yield for the process. Any future intensification in animal production within a pasture based system will demand an increase in the amount and the quality of forage supply. In this way, great amounts of nitrogen should be incorporated to the plant-soil system (Simpson and Stobbs, 1981). Basically, this can be achieved by periodical applications of nitrogen-containing fertilizers or, alternatively, through the symbiotic fixation of atmospheric nitrogen.

Legumes associate in a symbiotic way with bacteria belonging to the genus Rhizobium, thus producing root nodules where the nitrogen fixation process occurs. Recent agronomic studies about Lotus species cultivated in the introduction garden of the Instituto Tecnológico de Chascomús (INTECH), project L. pedunculatus and L. corniculatus var. hirsutus by their good adaptability to local conditions (Remis et al, 1996). The former is a commercial forage and the latter is a non commercial botanical variety. Both of them have still not been tested nor cultivated in the region. Nutritional evaluations done by our group (Locatelli et al., this issue) suggest that this two species might substantially improve the quality of pastures present in the zone by increasing their protein content.

Condensed tannins are known to be present in Lotus species and their compositions usually differ between them (Pankhurst and Jones, 1979; Pankhurst et al., 1987). These compounds could affect nodulation of such legumes and indirectly diminish plant persistence and quality, particularly in low fertile and poorly drained soils. Pankhurst and Jones (1979) found that delphinidin rich tannins accumulated in roots and ineffective nodules of L. pedunculatus. They suggested that such ineffectiveness could be due to the sensitivity of Rhizobium strains located in the nodules to those compounds. L. corniculatus var. hirsutus roots also contain a high level of delphinidin rich tannins, but lower than roots of L. pedunculatus plants. On the contrary, condensed tannins present in L. glaber do not contain delphinidin in their structure (Pankhurst and Jones, 1979; Pankhurst et al., 1987).

R. loti strains commonly found in soils of the Salado River Basin are able to form nodules in L. glaber roots. However, no data concerning the capacity of these strains to nodulate other Lotus species are currently available. The aim of the present work was to determine the ability of an indigenous strain of R. loti isolated from L. glaber to form effective nodules in other Lotus species. Data are compared with two additional strains of R. loti: one of them (NZP 2037) is known to form effective nodules in roots of both Lotus species used in this study, while the other (NZP 2213) forms ineffective nodules on some species which contain delphinidin rich tannins in their roots (e.g. L. pedunculatus) (Pankhurst et al., 1987).

MATERIALS AND METHODS

Plant material and culture: Legumes used in this study were L. corniculatus var. hirsutus and L. pedunculatus. Seeds obtained from the introduction garden were surface disinfected with concentrated sulfuric acid for 15 min, thoroughly rinsed with sterile distilled water and then kept in water for 24h. After that, they were placed in culture tubes containing 20 ml of solid Jensen medium (Vincent, 1970) and incubated in a climatized room at 25°C with a photoperiod of 16 h.

Rhizobium loti strains: Strain 1-INTECh was originally isolated from nodules of L. glaber plants naturally growing in the outskirts of the INTECH. Strains NZP 2037 and NZP 2213 from Beltsville Rhizobium Culture Collection were kindly gifted by Dr. Paul Beuselinck, USDA-ARS, University of Missouri, Columbia, MO, USA. Bacteria were cultured in AMA medium (Vincent, 1970), at 28°C with rotatory shaking.

Nodulation assay: Three days after transfer to culture tubes, seedlings were inoculated with 0.1 ml of bacterial culture and incubated in the above stated conditions for a 6 week period. Combinations of each Lotus species and bacterial strain were done, each one consisting of 24 plants. Control plants were inoculated with the same volume of AMA medium.

Nitrogenase activity determination: Nitrogenase activity of nodules was measured by using the acetylene reduction technique (Hardy et al., 1968) on the same plants used for the nodulation assay. Six weeks old plants were placed in 50 ml culture tubes (8 plants each) and hermetically closed with rubber caps. Each treatment included three replicate tubes. Air (5 ml) was withdrawn and replaced by an equivalent volume of acetylene. Tubes were incubated at 25°C during 2 h, and then 0.5 ml gas samples were removed and analysed for acetylene and ethylene content using a Hewlett-Packard 5890-A gas chromatograph fitted with a flame ionization detector and "Poropak N" in a 1.5 mm x 1.32 m column.

RESULTS

Figure 1 shows whole plants of L. pedunculatus (Fig. 1A) and L. corniculatus var. hirsutus (Fig. 1B) inoculated with three different strains of R. loti: the two collection strains NZP 2213 and NZP 2037, and the indigenous strain 1-INTECh. L. pedunculatus plants inoculated with strains NZP 2213 and 1-INTECh had a morphological aspect similar to that of non-inoculated plants (Fig. 1A). Their aerial parts were scarcely developed and exhibited chlorotic leaves. On the contrary, L. pedunculatus plants inoculated with strain NZP 2037 showed a normal development of aerial parts and no symptoms of chlorosis were detected on leaves. On the other hand, L. corniculatus var. hirsutus plants inoculated with all three strains grew more vigorously than controls (Fig. 1B), although some chlorosis was observed in leaves of plants inoculated with strain 1-INTECh. Nodules formed by strain NZP 2037 in both Lotus species looked similar . They were spherical and pink coloured. Strains NZP 2213 and 1-INTECh also formed this type of nodules in L. corniculatus var hirsutus plants but in L. pedunculatus plants they formed pseudonodules.

Figure 1: Nodulation of plantlets of L. pedunculatus (A) and L. corniculatus var hirsutus (B) by Rhizobium strains (from left to right : control, 1-INTECh, NZP 2213 and NZP 2037).

Table 1 shows results obtained when nitrogenase activity was measured in nodules of Lotus plants inoculated with the three different R. loti strains. As shown, strain NZP 2037 was capable of forming effective, nitrogen-fixing (Nod+ fix+) nodules with the two species of Lotus. Strain NZP 2213 formed (Nod+ Fix+) nodules in L. corniculatus var. hirsutus and ineffective (Nod+ Fix-) nodules in L. pedunculatus. Indigenous strain 1-INTECh formed (Nod+ Fix-) nodules in both species, even when the morphology of those formed in L. corniculatus var. hirsutus was very similar to that of effective ones.

Table 1. Atmospheric nitrogen fixing ability of root nodules of Lotus species inoculated with three different R. loti strains.
Nitrogenase activity (pmol ethylene/ h / plant)
Rhizobium strain
L. pedunculatus
L. corniculatus var. hirsutus
None
0.80 ± 0.06
1.18 ± 0.07
NZP 2037
12.00 ± 0.13
6.30 ± 0.52
NZP 2213
0.70 ± 0.05
8.84 ± 0.42
1-INTECh
1.37 ± 0.15
1.66 ± 0.51

DISCUSSION

The observed ability of the R. loti collection strains NZP 2213 and NZP 2037 to nodulate L. pedunculatus and L. corniculatus var. hirsutus, as well as efectiveness of such nodules is in agreement with results reported by Pankhurst et al. (1987), except for the interaction between NZP 2213 and L. corniculatus var. hirsutus. In our hands, the latter symbiosis association rendered effective nodules (Nod+ fix+), which is in contrast with previous results reported by Pankhurst el al. (1987) showing the formation of ineffective nodules. A possible explanation for this discrepancy could be attributed to physiological changes in L. corniculatus var. hirsutus performed after adaption to soils of our region.

Concerning the inability of the indigenous R. loti strain 1-INTECh to form effective nodules in roots of both Lotus species studied, it is concluded that Rhizobium strains capable of forming effective nodules and succesfully compete with native strains should be obtained to introduce these plant species. Thus, if future attempts to cultivate L. pedunculatus or L. corniculatus var. hirsutus are made in the region, the use of such adequate bacterial strains could greatly contribute to the improvement of legume forage productivity and quality. However, it is necessary to point out that these experiments should be checked by using a greater number of indigenous isolates, to confirm that the behavior of strain 1-INTECh is representative of the local R. loti population.

ACKNOWLEDGMENTS

The authors wish to thank M.L. Locatelli for helpful discussion. MJE is a Fellow from Comisión de Investigaciones Científicas (CIC, Bs. As.). RAU and AAI are Members of the Investigator Career from Consejo Nacional de Investigaciones Científicas (CONICET).

REFERENCES.

Hardy, R.W.F.; R.D. Holsten; E.K. Jackson and R.C. Burns (1988) Plant Physiol. 43: 1185-1207.

Pankhurst, C.E. and W.T. Jones (1979) J. Exp. Bot. 30: 1109-1118.

Pankhurst, C.E.; D.H. Hopcroft and W.T. Jones (1987) Can. J. Bot. 65: 2676-2685

Remis, J.L.; O.A.Ruiz; R.A.Ugalde and A.A.Iglesias (1995) Lotus Newsletter 26:17-20

Simpson, J.R. and T.H. Stobbs (1981) In: Grazing Animals (Morley, F.W., ed.). pp. 261-287. Elsevier Scientific Publishing Company, Melbourne.

Vincent, J.M. (1970) A Manual for the Practical Study of Root Nodule Bacteria. Blackwell Scientific Publications, Oxford.


EVALUATION OF FORAGE QUALITY ATTRIBUTES FOR LOTUS SPP. GROWN IN THE SALADO RIVER BASIN

M. Laura Locatelli, José L. Remis and Alberto A. Iglesias.

Instituto Tecnológico de Chascomús (INTECH - CONICET)
Camino Circunv. Laguna
km 6. Casilla de Correo 164
Chascomús (7130). Bs. As. Argentina.

INTRODUCTION

Beef and dairy cattle production in the Salado River Basin is essentially a grazing system mainly based on natural grasslands, with a minor portion of cultivated pastures. Edaphic characteristics: limed and poorly drained soils with severe phosphorus deficiency, medium organic matter levels, high alkalinity and salinity; together with periodic exposition of soils to waterlogged conditions, significantly decrease persistence and yield of common legume species (red clover, alfalfa, white clover) in this region (Mazzanti et al., 1986). A strategy to be developed to solve these problems is the introduction of foreign legume species. In this way, Lotus spp. is an important alternative, after their growing conditions and significant nutritional value. Currently, the genus Lotus has been introduced in the Salado River Basin which counts with L. corniculatus and L. glaber (already naturalized) species. However, at the present time, information about nutritional quality (and its variability throughout the year) of commercial species in the area is scarce, and an exhaustive analysis on the productive performance of Lotus in the region is far from complete.

In grazing systems (preferentially extensive) animal production will be highly influenced by nutritional variability of forage offer along the year. Therefore, it is relevant to know (besides biomass production) the nutritional attributes (protein, energy, etc.) and the efficiency by which ruminants profit different legume forage species. After this information, it is possible to design different utilization and agronomic management strategies to obtain a maximum yield from a specific forage material. It has been reported (McGraw et al., 1989) the relevance of an early evaluation of quality parameters in introduction trials of unknown species, in order to identify desirable or undesirable characteristics and thus to postulate rationale strategies for the improvement of forage quality.

Last year, we reported studies carried out at Instituto Tecnológico de Chascomús (INTECH) concerning green and dry matter production for different Lotus spp. growing at an introduction garden located in the Salado River Basin region (Remis et al., 1995). The present study completes that previous evaluation by analysis of chemical and biological parameters determining the quality of the different plant species.

MATERIALS AND METHODS

Aerial biomass (whole plant) of the different Lotus entries (perennial species) was sampled from small plots of the introduction garden during years 1994 and 1995. According to seasonal periods and plant species, these samples included leaves, stems, flowers and/or fruits. Distribution in the different seasonal periods was made by considering the time of sample collection as follows: i) Fall-winter: from late March to early August, ii) Spring: from mid October to mid November, and iii) Summer: from late December to mid February. Samples were oven-dried at 60°C until constant weight to determinate dry matter (DM) content (data expressed as % of the aerial biomass), and then milled to 1 mm by using a Wiley-type mill.

Crude protein (CP) was estimated in the samples by total nitrogen determination after sulfuric acid digestion, distillation and titration by the Kjeldahl method (N x 6.25). Neutral detergent fiber (NDF) was determined according to Goering and Van Soest (1970). The enzymatic pepsin-cellulase method (Donofer et al., 1963) was use to measure DM in vitro digestibility (DMIVD) of the samples. CP, NDF and DMIVD are expressed as % of the DM.

RESULTS AND DISCUSSION

Table 1 shows quality parameters determined for periods of spring and summer in different Lotus spp. grown at the introduction garden in INTECH. Data corresponding to Medicago sativa and Trifolium pratense are included to establish a comparison with two commonly used forage species. As shown (Table 1) besides L. glaber and L. corniculatus (the two commercial species already used in the region) other forage materials exhibited relatively low NDF levels, as well as CP and DMIVD values higher than the mean found for the different Lotus spp. analyzed. In this way, L. corniculatus var. hirsutus showed a good pattern of quality parameters, thus reinforcing the promising data previously obtained with this species (Remis et al., 1995). Table 1 also shows that the hybrid L. corniculatus x L. pedunculatus exhibited high values of CP and DMIVD during the spring, whereas similar results are observed for L. parviflorus during the summer peirod. Concerning L. rectus, Table 1 shows that this species exhibited low parameter values estimating quality. However, it needs to be considered that L. rectus was the only arbustive species in the introduction garden, and that the data could be masked by a significant proportion of lignified stems in the samples. Periodic defoliations of aerial biomass could reverse the low leaf to stem ratio found in our samples and renders a more actual picture of the nutritive value of L. rectus when utilized by ruminants.

Results obtained with L. corniculatus and L. pedunculatus are in agreement with those reported by Carámbula et al. (1994) for acid, poorly drained, and phosphorus depleted soils in the East of Uruguay. Concerning the latter species, Table 1 shows relatively high levels of CP content but low DMIVD values. It is necessary to point out that the methodology we applied in this study determines only ruminal digestibility. Since L. pedunculatus species possesses high condensed tannin levels (5-10% of the DM) (Lowther et al., 1987; Roberts and Beuselinck, 1992), it is feasible that these compounds decrease DM digestibility at rumen (Chiquette et al., 1989; Waghorn et al., 1994), although DM digestion of this species at hind gut could equilibrate the global process (Barry et al., 1986).

As shown in Figure 1, quality parameters for L. corniculatus var. hirsutus are maintained relatively high along the year, being the variations comparable with those observed in L. corniculatus and L. glaber. It has been reported (Forde and de Lautour, 1978; Kelman and Tanner, 1990) that L. corniculatus var. hirsutus contents higher amounts of condensed tannins (3.5-7% of the DM) than L. corniculatus and L. glaber, which determines a nutritional advantage for the former species. Taking into account these results, and considering the reseed capacity and high yields of DM found for L. corniculatus var. hirsutus at the introduction garden (Remis et al., 1995), together with the high quality nutritional characteristics reported herein; it is concluded that this species is potentially important for the region. Future incorporation of L. corniculatus var hirsutus into forage mixed pastures or through interseed on natural grasslands, as well as its use to obtain hybrid species possessing higher productivity and quality are important alternatives to be developed for the improvement of pastures in the region. Works in our Institute are on the way, looking for technological tools to reach an increase of productivity in the Salado River Basin.

ACKNOWLEDGMENTS

The authors are greatly indebted to D.F. Gómez Casati and G.A. Polenta for helpful suggestions on the experimental procedures. AAI is a Member of the Investigator Career from CONICET.

REFERENCES

Barry, T.N., Manley, T.R. and Duncan, S.J. 1986. The role of condensed tannins in the nutritional value of Lotus pedunculatus for sheep. 4. Sites of carbohydrates and protein digestion as influenced by dietary reactive tannin concentration. British J. Nutr. 55:123-137.

Carámbula, M., Ayala, W. and Carriquiry, E. 1994. Lotus pedunculatus. Adelantos sobre una forrajera que promete. Serie Técnica Nº 45. INIA, Uruguay.

Chiquette, J., Cheng, K.J., Rode, L.M. and Milligan, L.P. 1989. Effect of tannin content in the two isosynthetic strains of birdsfoot trefoil (Lotus corniculatus L.) on feed, digestibility and rumen fluid composition in sheep. Can. J. Anim. Sci. 69:1031-1039.

Donefer, E. Niemann, P.J., Crampton, E.W. and Lloyd, L.E. 1963. Dry matter disappearance by enzyme and aqueous solutions to predict the nutritive value of forages. J. Dairy Sci. 46:965-970.

Forde, M.B. and de Latour, G. 1978. Plant introduction trials. Classification of Lotus introductions. N.Z. J. Exptl. Agr. 6:293-7.

Goering, H.K. and Van Soest, P.J. 1970. Forage fiber analysis (apparatus, reagents, procedures and some applications). Agric. Handbook 379. USDA.

Kelman, W.M. and Tanner, G.J. 1990. Foliar condensed tannin levels in Lotus species growing on limed and unlimed soils in South-Eastern Australia. Proc. N.Z. Grassl. Assoc. 52:51-54.

Lowther, W.L., Manley, T.R. and Barry, T.N. 1987. Condensed tannin concentrations in Lotus pedunculatus cultivars grown under low soil fertility conditions. N.Z. J. Agric. Res. 30:23-25.

Mazzanti, A., Darwich, N.A., Cheppi, C. and Sarlangue, H. 1986. Persistencia de pasturas cultivadas en zonas ganaderas de la Pcia. de Buenos Aires. Rev. Arg. Prod. Anim. 6 (Sup. 1):65.

McGraw, R.L., Beuselinck, P.R. and Marten, G.C. 1989. Agronomic and forage quality attributes of diverse entries of birdsfoot trefoil. Crop Sci. 29:1160-1164.

Remis, J.L., Ruíz, O.A., Ugalde, R.A. and Iglesias, A.A. 1995. Evaluation of Lotus spp. growth in the Salado River Basin. Lotus Newslett. 26:17-20.

Roberts, C.A. and Beuselinck, P.R. 1992. Condensed tannins in Lotus species. Lotus Newslett. 23:41.

Waghorn, G.C., Shelton, I.D. and McNabb, W.C. 1994. Effects of condensed tannins in Lotus pedunculatus on its nutritive value for sheep. 1. Non-nitrogenous aspects. J. Agric. Sci. 123:99-107.

Table 1: Nutritive parameters (%) in whole plants of Lotus spp. during spring-summer periods.
Spring
Summer
Species
DM
NDF
DMIVD
CP
DM
NDF
DMIVD
CP
M. sativa1
24.2
46.0
65.6
16.5
24.2
47.0
62.1
16.8
T. pratense1
18.0
38.0
65.0
21.0
24.0
44.0
57.0
15.0
L. glaber
24.0
26.8
71.5
24.9
30.0
48.6
55.9
14.4
L. corniculatus
22.0
37.2
63.1
18.6
25.0
52.1
49.3
10.9
L. corniculatus var. hirsutus
19.7
33.7
58.1
19.5
24.0
47.4
55.4
14.0
L. corniculatus var. alpinus
18.0
52.0
49.1
14.1
L. corniculatus x pedunculatus
15.5
34.2
64.7
21.9
30.6
44.6
53.4
10.3
L. filicaulis
23.2
54.2
41.6
10.1
L. parviflorus
31.4
45.0
57.6
15.0
L. pedunculatus
16.4
43.3
52.5
18.7
33.0
43.8
18.5
L. rectus
21.2
55.5
46.1
14.2
29.0
51.8
45.6
15.5
MEAN
19.8
38.5
59.3
19.6
27.1
49.5
50.2
13.6
DEVIATION
3.3
9.9
9.1
3.6
4.9
3.6
5.7
2.8

1 Data from Fernández and Galli. 1993 (INTA Rafaela, Argentina) corresponding to studies carried out in the humid pampa region (Argentina).


Figure 1: Seasonal variation for quality parameters obtained from whole plants of L. glaber (continuous lines), L. corniculatus (big dashes) and L. corniculatus var. hirsutus (small dashes). Spg = spring; Sum = summer; and F/W = fall/winter.


ALTERATIONS OBSERVED IN LOTUS GLABER (SYN. L. TENUIS) SEEDLINGS APPARENTLY INDUCED BY ETHEPHON

Mujica MM1 and CP Rumi2

1Area de Genética y Mejoramiento Animal y Vegetal
2Instituto de Fisiología Vegetal
Facultad de Ciencias Agrarias y Forestales, UNLP
CC 31 (1900) La Plata, Argentina

Ethephon (2-chloroalkylphosphonic acid) is regarded as "liquid" ethylene and is probably widely used as plant growth regulator in agriculture. Ethephon is an ethylene generator. Their breakdown occurs primarily on the leaf surface and in the aqueous plants fluids (Abeles et al. 1992). The ethylene acting as a natural volatile hormone and have an effect on many physiological process.

The objective of this communication was to inform about some abnormalities registered by the seedlings growth of Lotus glaber, presumably due to an accidental exposition to exogenous ethephon.

L. glaber seeds were sown in vermiculite contained in a tray. They were cultivated in a greenhouse with a supply of nitrofoska 2 (BASF) 3gr/l. The substrate was maintained at the water saturation point. From their sowing this tray remained accidentally exposed to an ethephon sprinkled (10-4 a 10-5 M) applied on another trial.

After the seedling emergence it was observed a notorius alteration in the uniformity size of the seedlings aerial part. Among the fourth and fifth weeks the seedlings were removed from the tray and it were evaluated the roots and the aerial components.

The seedling abnormalities observed were: The roots of the most seedlings showed some alterations in their morphology and size, inhibition of roots elongation coupled with lateral swelling , hypertrophyes, be bent into curls and a negative gravitropism in lateral roots. The more important seedling abnormalities are shown in the figure 1 (a). Seedlings with their roots affected and also normal seedlings were registered, (n=114). The frequency of seedlings affected and unaffected was 73.68% and 26.32%, respectively.

Figure 1. (a) Seedling abnormalities: small size of aerial part, hypertrophyes and curls in the root system. (b) Recovery of the abnormal seedlings after fourth weeks cultivated at the open air.

The abnormal plants were cultivated during fourth weeks at the open air in pots with agricultural ground to evaluate their following behavior. During the fourth weeks that the plants were cultivated in the open air it was register that the affected plants were recovering gradually their normal growth. Immediately to the end of this period was observed that, from the affected roots had been originated roots with normal morphology (figure 1 ( b)).

REFERENCES

Abeles FB, PW Morgan and ME Saltveit Jr (1992) Ethylene plant biology. Academic Press Inc.. New York. 414 pp.


NEW MORPHOLOGICAL PHASES DELIMIT LOTUS STRIGOSUS (NUTTALL) GREENE VAR. TOMENTELLUS ISELY FROM LOWER CALIFORNIA AND SONORA, MEXICO (FABACEAE -- LOTEAE)

Ana M. Arambarri

Area de Botánica, Jardín Botánico y Arboretum "C. Spegazzini"
Facultad de Cs. Agrarias y Ftales - UNLP
C. C. 31, 1900 La Plata, Argentina

Abstract. The Lotus strigosus var. tomentellus complex from Lower California and Sonora, Mexico in which "phases" to denote aberrant forms or intermediates within a species have been found was studied morphologically. Until present the phases 1 and 2 are knowledge for L. strigosus var. tomentellus Isely. The specimens of herbarium (MEXU) were examined with a stereoscopic microscope and the topographic features of the testa were analyzed by SEM. According to morphological characteristics found the specimens studied were separated in three groups. The group 1 correspond to L. strigosus var. tomentellus "phase 1" Isely. It is characterized by the presence of peduncled inflorescence with flowers more than 7 mm length, and the bract 1-2 (-3) leaflets. The groups 2 and 3 agree with L. strigosus var. tomentellus Isely by the presence of flowers no more than 6 mm length, subsessiles or shortly peduncled and bract absent. However, fruit and seed characteristics delimit two new morphological phases to L. strigosus var. tomentellus from Lower California and Sonora, Mexico. They are "phase 3" to group 2 with fruit incurved, falcate, and seed with the hilum superficial, and "phase 4" to group 3 with fruit distally incurved and seed with the hilum sunken in a lateral notch.

Key words: Fabaceae; Loteae; Lotus; Lower California; Mexico; morphology; phases; Sonora.

Introduction

Isely (1981) uses the word "phases" to denote aberrant forms or intermediates within a species. Such "phases" have been found in this study in the L. strigosus var. tomentellus complex from Lower California and Sonora, Mexico.

Lotus strigosus (Nutt.) Greene, L. strigosus var. hirtellus (Greene) Ottley and L. strigosus var. tomentellus (Greene) Isely are distributed in California, Lower California, and Mexico.

The Lotus strigosus group shows a continuous variation in its morphological characteristics. According to Isely (1981), Nuttall's (1838) names Hosackia nudiflora, H. rubella and H. strigosa illustrate local population variance. Greene (1890) examined and subdivided Nuttall's species in the L. strigosus group. He renamed L. tomentellus to one species from Lower California.

Later, Ottley (1923, 1944) distinguished three taxa, namely, L. strigosus, L. strigosus var. hirtellus and L. tomentellus which she based on seed characteristics. She described the seed of L. strigosus, and L. strigosus var. hirtellus as: "cubical, notched at the hilum" and that of L. tomentellus as "globose to oval an occasional one cubical."

Isely (1981) reduced L. tomentellus to a variety of L. strigosus. At the same time he established two phases, both for L. strigosus (phase 1 and 2) and for the var. tomentellus (phase 1 and 2) based on the fact that the phases represent "aberrant forms or intermediates."

This study redefines the characteristics found in specimens of L. strigosus var. tomentellus from Lower California and Sonora.

Materials and Methods

The specimens studied were from (MEXU), Herbario Nacional de Mexico, Instituto de Biologia, Universidad Nacional Autónoma de Mexico. The acronym for the herbarium is given according to Holmgren et al. (1990). The morphological characters were examined with a Wild M 8 stereoscopic microscope equipped with a camera lucid. The topographic features of the testa were examined from whole seeds and portions of them by means of a Jeol JSM T100 scanning electron microscope (SEM). Drawings of the characters were prepared.

Results and Discussion

According to morphological characteristics, the specimens studied have been separated in groups as follows:

Group 1.

LOWER CALIFORNIA. Western base of Cocupah Mts., on shores of Laguna Maguata, C. Epling, M. Darsie, W. M. Stewart and W. M. Robison, s. n. Feb-19-1933 (552731 MEXU).- Loc. Puerto Refugio, Punta Norte de la Isla Angel de la Guarda, Mpio. Ensenada, 029º 33' 00 N lat, 113º 34' 00 W long, Elev. 50 m s. n. m. Pedro Tenorio L. 10849, C. Romero de T., Feb-07-1986. Desierto sarcocaulescente, suelo arenoso-pedregoso (523662 MEXU).

SONORA. Tiburon Island, Ensenada Blanca, vicinity 28º 59' N lat, 112º 29½' W long, Joe Edmundson; Alexander Russell, R. S. Felger , R.S.F. 17276, Feb-20-1968. (447029 MEXU).

Group 2.

LOWER CALIFORNIA. Loc. Desierto de Vizcaino, Arroyo San José de Castro, Mpio. Mulejé. Elev. 160 m s. n. m.; Jorge Cancino Hernández 6, Jun-26-1983. Hierba anual de 7 cm . Abundancia regular. Vegetación circundante matorral subinerme, vegetación halófila, en potrero. (354700 MEXU).- Loc. Desierto de Vizcaino, E de Bahía Asunción, Mpio. Mulegé. Jorge Cancino, Carlos Plata, C. 68; Feb-11-1984. Hierba anual escasa, 10 - 15 cm altura. Vegetación primaria de ecotono, duna mat. halófilo; suelo arenoso. (428089 MEXU).- Loc. La Bocana, Mpio. Santa Rosalía, 26º 53' N lat, 113º 44' W long. Elev. 20 m s. n. m. Pedro Tenorio L 12927, C. Romero T. Apr-17-1987. Hierba escasa, flores amarillas. Vegetación de dunas; suelo amarillo arenoso. [(282843 MEXU), both L. strigosus var. tomentellus and L. salsuginosus var. brevivexillus are on the same sheet].

SONORA. Tiburon Island, Ensenada de La Perra; vicinity 28º 47' N lat, 112º 16' long. R. S. Felger, J. Edmundson, N. Thomas, R. S. F. 17724, Apr-12-1968. (453698 MEXU).

Group 3.

LOWER CALIFORNIA. Loc. San Ignacito, 14 km al SW de Cataviña, por la carr. transpeninsular, Mpio. Ensenada. 29º 47' N lat, 114º 45' W long. Elev. 640 m s. n. m. Pedro Tenorio L. 13110, C. Romero de T. Apr-28-1987. Hierba postrada, abundante, fl. amarilla, fr. inmaduro. Vegetación matorral desértico, primaria; suelo arenoso amarillo. [(155141 MEXU), specimen corresponds to L. strigosus var. tomentellus, the envelope attached contains a mixture of the specimen L. strigosus var. tomentellus with L. salsuginosus var. brevivexillus].

SONORA. On hillsides of volcanic rock 2.5-3 mi N of Sáric, approx. 31º 07' N lat, 111º 20' W long. Elev. 2900 ft.; Grady L. Webster 22517, Mar-26-1978. Scrub of mesquite, saguaro, Cercidium, et al. Postrate, fls. yellow. (305643 MEXU).- Coast of the Gulf of California near the mouth of the Rio Concepcion, 10.6 mi. NE of El Desemboque on the road toward Caborca. Near 30º 40' N lat, 112º 57' W long. Elev. 100 ft. A. C. Sanders, M. Dimmitt, G. Montgomery, et al. 3479, Mar-7-1983. Sandy coastal flats with low dunes; creosote Bush Scrub with Larrea and Ambrosia dunosa. A fairly common yellow-flowered annual. (358773 MEXU).- Loc. km 71 de la carr. Sonoyta-San Luis Río Colorado. 32º 04' N lat, 113º 34' W long. Alvaro Campos V 4435a, J. L. Panero, L. I. Cabrera, Feb-28-1992. Hierba 20 cm altura, fl. blanca, fr. verde, frecuente. Vegetación matorral espinoso con Opuntia. (578101 MEXU).

Group 2 fruit incurved, falcate and seed with the hilum superficial.

Group 3 fruit distally incurved and seed with the hilum sunken in a lateral notch.

KEY TO "PHASES" OF L. STRIGOSUS VAR. TOMENTELLUS FROM LOWER CALIFORNIA AND SONORA, MEXICO

Acknowledgments

I express my appreciation to Curators of the Herbario Nacional de Mexico (MEXU). I also thank Prof. William F. Grant for his helpful comments on the manuscript, and Rafael Urrejola for technical assistance using SEM.

References

Greene, E. L. 1890. Enumeration of the North American Loti. Pittonia 2: 133-150.

Holmgren, P. K. , N. H. Holmgren and L. C. Barnett. 1990. Index Herbariorum. P. 1. The Herbaria of the World. [Regnum Veg. 120]. NY Bot. Gard., Bronx.

Isely , D. 1981. Leguminosae of the United States. III. Subfamily Papilionoideae: tribes Sophoreae, Podalyrieae, Loteae. Mem. NY Bot. Gard. 29(3): 124-264.

Nuttall, T., Torrey, J. and A. Gray. 1838. Leguminosae. In: Flora of North America 1: 326. NY.

Ottley, A. M. 1923. A revision of the Californian species of Lotus. Univ. Calif. Publ. Bot. 10: 189-305.

Ottley, A. M. 1944. The American Loti with special consideration of a proposed new section, Simpeteria. Brittonia 5(2): 81-123.


PRODUCTION AND SOIL ENHANCING LEGUME LIVING MULCH SYSTEM FOR AGRONOMIC CROPS

N. L. Hartwig

Prof. of Weed Sci., Dept. of Agronomy, Pennsylvania State University, University Park, PA. 16802

ABSTRACT

Crownvetch (Coronilla varia L.) and birdsfoot trefoil (Lotus corniculatus L.) can be used as perennial living mulches in the production of corn (Zea mays L.), soybeans (Glycine max L.) small grains and forages. The primary benefit is the almost total reduction in soil erosion. Other benefits are enhanced productivity through soil conservation, increased soil organic matter and organic nitrogen, improved soil tilth, better footing during spring planting and fall harvesting seasons when it might otherwise be too wet to get into the field. Positive side effects include a reduction in environmental pollution from soil, nutrient and pesticide runoff, recycling of nutrients from deeper in the soil profile and more flexibility in meeting soil erosion criteria, including increasing the amount of corn and soybeans that can be grown on sloping cropland than otherwise would be allowed.

With the recent introduction of imidazolinone resistant (IR) corn and a weed control program based on imazethapyr, it is now possible to establish these legumes in corn without injuring the corn or the legume ground cover. Crownvetch is very slow to establish but when once established it will persist forever with the right management. Birdsfoot trefoil provides quick cover but gradually thins out over a period of years as a result of crownvetch competition and chemical suppression, leaving a predominately crownvetch cover. Corn of any kind can be planted into a one year old stand of birdsfoot trefoil and crownvetch using a recommended rate of rimsulfuron + thifensulfuron (Basis) for weed control with little or no injury to the cover crop or corn. Atrazine at a rate of 0.5 to 0.75 lb/A needs to be tank mixed with Basis to suppress the birdsfoot trefoil so it won't compete with the corn. Generally corn yields have not been suppressed when cover crop suppression is 95 to 98% for the first six weeks after planting. Fortunately crownvetch and to some extent birdsfoot trefoil will recover and provide up to 80% ground cover by fall even after suppression of this magnitude early in the summer.

Birdsfoot trefoil that is one year old and crownvetch that is two or more years old will tolerate 0.5 lb/A of glyphosate applied at about the time dandelion (Taraxacum officinale Weber in Wiggers) is in bloom. This makes it possible to plant Roundup Ready¨ soybeans and use low rates of glyphosate for weed control and crownvetch or birdsfoot trefoil suppression. Roundup Ready¨ soybean yields up to 60 bu/A were obtained when planted into crownvetch in 1996 but yields up to 75 bu/A were obtained where there was no cover crop. It would appear that some refinement of this system may be necessary to prevent soybean yield suppression when planting into a crownvetch or birdsfoot trefoil living mulch.

Small grains and alfalfa have also been planted into crownvetch and grown without a loss in yield and without losing the cover crop. A blanket treatment of glyphosate or paraquat is commonly necessary before planting small grains or alfalfa to suppress the cover crop for the first six weeks of crop growth. Post treatments may be necessary for annual or winter annual weed control. Two herbicides that crownvetch will not tolerate are clopyralid and 2,4-DB but it will tolerate recommended rates of most other small grain or alfalfa herbicides and still be there in sufficient amounts so reseeding is not necessary when rotating back to corn.

The whole program is explained on the web at: Living Mulch


PRELIMINARY ISOZYME DATA FOR LOTUS GLABER (SYN. L. TENUIS) AND LOTUS CORNICULATUS

Esteves, P.; Kade, M. and O.H. Caso
Centro de Ecofisiología Vegetal (CONICET)
Serrano 669
1414 Buenos Aires. Argentina

Two species of the genus Lotus are agriculturally important in Argentina because they are adapted to harsh environmental conditions marginal for alfalfa production. Lotus corniculatus L. is the main forage legume in Entre Ríos Province (Vertisol soils) and L. glaber Mill. has become naturalized along the Flooding Pampa (Molisols and Alfisols).

In spite of the mentioned characteristics practically no breeding efforts have been performed on these species in Argentina in order to increase their productivity and persistence.

The basis of any breeding program is to identify and quantify the species variability (Brown, 1978). To manage this, one possibility is to characterize isozyme polymorphisms and their distribution within and among populations (Tanksley and Orton, 1983; Ibañez et al., 1993). Indeed, isozyme patterns were used to elucidate the origin of L. corniculatus (Raelson and Grant, 1988). It was proposed that L. corniculatus is an allotetraploid being probably L. glaber and/or L. alpinus the maternal parents of the original hybrid (Ross and Jones, 1985). To our knowledge, no such studies have been performed in L. glaber.

In this context, preliminary results of isozyme polymorphisms were investigated, including other enzyme systems, in order to assess the extent of isozyme variability among and within the Argentine cultivars of L. glaber and L. corniculatus.

Experimental procedures

Plant material: Commercial seeds of L. glaber (2n=2x=12) cv. Tresur Chajá and L. corniculatus (2n=4x=24) cv. El Boyero INTA were subjected to chromosome number determination and germinated following ISTA Rules (1985). When seedlings developed the first leaves, they were transferred to greenhouse conditions (15-25C) during 60 days.

Isozyme electrophoresis: To obtain crude extracts, leaf material was crushed thoroughly in chilled mortars with cold extraction buffer (1:1,5 w:v). Each sample consisted of young leaflets of at least 5 plants for each species. Experiments were replicated twice with similar results. Extracts were absorbed onto 10 x 5 mm wicks of filter paper (Whatman No. 3) and inmediately inserted into slots performed in the starch gel. Electrophoresis was carried out using 11% starch (Sigma) gels prepared with a tris-citrate pH 7.0 buffer for malate dehydrogenase (MDH), 6-phosphogluconate (6-PG) and aspartate aminotranspherase (AAT), tris-citrate/lithium borate pH 8.3 buffer for esterase (EST) and diaphorase (DIA) and histidine/citric acid pH 5.7 buffer for acid phosphatase (ACP). These methods and staining procedures to resolve isozymes are described elsewhere ( Scandalios and Sorensen, 1977).

Gels were incubated at 37C for one hour, in the dark; then they were fixed in methanol 50%.

Results

The enzymes characterized displayed different patterns for each cultivar analyzed, with the exception of AAT and 6-PG. There was an apparent monomorphism among and within L. glaber and L. corniculatus cultivars for these isozymes.

MDH produced different electrophoretic patterns for each species showing the greatest variability inside the cultivars evaluated. Lotus glaber exhibited three patterns, which had two or three bands in different possitions. That pattern composed of two bands was also found in some L. corniculatus individuals. L. glaber samples consisted of one or three bands resulting in four different patterns.

EST showed two isozyme patterns in each species: L. glaber samples produced a third esterase band not found among L. corniculatus patterns, but both cultivars shared the pattern composed of two variable bands.

DIA enzymes were only evaluated in L. glaber and two different electrophoretic patterns were characterized consisting of two bands each.

Patterns of the ACP system provided the greatest differentiation between species. The cathodal region of the ACP gels in both species showed a common band. However, in the anodal region L. glaber ACP bands were almost always faster than L. corniculatus bands.

Discussion

Differences in electrophoretic patterns of isozymes for cv. Tresur Chajá (L. glaber) and cv. El Boyero INTA (L. corniculatus) were detected between and among cultivars. MDH, EST and ACP systems showed some same patterns. Such a finding may be related to the presumed hybrid ancestry of L. corniculatus. Ross and Jones (1985) suggested that either L. alpinus and/or L. glaber could be the maternal parent of the original hybrid.

Further studies would confirm if ACP system can be used effectively to differentiate one cultivar from another. The fast band for L. glaber pattern showed a higher mobility than the same band for L. corniculatus. This difference was not observed to be constant, but it is evident from this survey that its frequency is high. It is clear that these results need to be confirmed with a larger set of individuals, and perhaps, with small refinements of the procedure. Besides, Raelson and Grant (1988) did not examined ACP system in their review concerning the origin of L. corniculatus.

Electrophoretic analysis, which can be also performed on ungerminated seeds, would resolve questions of seed identification. L. glaber and L. corniculatus seeds are very similar, therefore a relatively rapid and accurate method of discriminartion, as well as the flavonol analysis based on a chromatographic procedure (Kade et al., 1997), would facilitate classification of seed samples.

References

Brown, A.H.D. 1978. Theor Appl Genet, 52: 145-157.

Ibañez, M.A., Di Renzo, M.A. and M.M. Proverene. 1993. Scientia Horticulturae 53: 281-288.

International Rules for Seed Testing. Rules 1985. 1985. Seed Sci. and Technol. 13: 299-355.

Kade, M., Wagner, M.L., Strittmatter, C.D., Ricco, R.A. and A.A.Gurni. 1997. Seed Sci. and Technol.. (in press).

Raelson, J.V. and W.F. Grant. 1988. Theor Appl Genet 76:267-276.

Ross, M.D. and W.T. Jones. 1985. Theor Appl Genet 71: 284-285.

Scandalios, J.G. and J.C. Sorensen. 1977. 1. Isozymes in Plant Tissue Culture. In: Plant Cell, Tissue and Organ Culture. Chapter VII, pp. 719. Ed. by J. Reinert and P.S. Bajaj. Springer Verlag.

Tanksley, S.D. and T.J. Orton. 1983. Isozymes in Plant Genetics and Breeding. Part A, B. Elsevier, Amsterdam.


THE INTERNATIONAL ASSOCIATION FOR PLANT TAXONOMY (IAPT) ANNOUNCES:
REGISTRATION OF PLANT NAMES

International Association for Plant Taxonomy
c/o Botanischer Garten & Botanisches Museum Berlin-Dahlem
Koenigin-Luise-Str. 6-8
D-14191 Berlin, Germany.

Test and Trial Phase (1998-1999)
Introduction
Subject to ratification by the XVI International Botanical Congress (St Louis, 1999) of a rule already included in the International code of botanical nomenclature (Art. 32.1-2 of the Tokyo Code), new names of plants and fungi will have to be registered in order to be validly published after the 1st of January 2000. To demonstrate feasibility of a registration system, the International Association for Plant Taxonomy (IAPT) undertakes a trial of registration, on a non-mandatory basis, for a two-years period starting 1 January 1998. The co-ordinating centre will be the Secretariat of IAPT, currently at the Botanic Garden and Botanical Museum Berlin-Dahlem, Germany. Co-ordination with present indexing centres for major groups of plants is being sought, in view of their possible active involvement at the implementation stage. The International Mycological Institute in Egham, U. K., has already accepted to act as associate registration centre for the whole of fungi, including fossil fungi.

Registration procedure
The co-ordinating registration centre (IAPT Secretariat), and any associated centre operating under its auspices, will register and make available all names of new taxa, all new combinations or rank transfers that are brought to their attention in one of the following ways:
by being published in an accredited journal or serial;
by being submitted for registration (normally by the author or one of the authors), either directly or through a national registration office; or
(for the non-mandatory trial phase only) as a result of scanning of other published information by the registration centres' own staff.

Registration by way of publication in accredited journals or serials

For a journal or serial to be accredited, its publishers must commit themselves, by a signed agreement with the IAPT, to
point out any nomenclatural novelties in each individual issue of their journal or serial, either by including a separate index of novelties or in another suitable, previously agreed way;
submit each individual issue, as soon as published and by the most rapid way, to a pre-defined registration office or centre.

Accredited journals and serials will be entitled, and even encouraged, to mention that accreditation on their cover, title page or in their impressum.

A permanently updated list of accredited journals and serials is being placed on the World Wide Web (http://www.bgbm.fu-berlin.de/iapt/registration/journals.htm). This list will be published annually in the journal Taxon.

Registration by way of submission to registration offices
Authors of botanical nomenclatural novelties that do not appear in an accredited journal or serial (but e.g. in a monograph, pamphlet, or non-accredited periodical publication) are strongly encouraged to submit their names for registration - and will be required to do so once registration becomes mandatory - in the following way:
all names to be registered are to be listed on an appropriate registration form, using a separate form for each separate publication;
the form (in triplicate) must be submitted together with two copies of the publication itself, either to a national registration office (see below) or, optionally, directly to the appropriate registration centre. Reprints of articles from books or non-accredited periodicals are acceptable, provided their source is stated accurately and in full;
one dated copy of each form will be sent back to the submitting author in acknowledgement of effected registration.

Registration forms can be obtained free of charge (a) by sending a request to any registration office or centre, by letter, fax or e-mail, or (b), preferably, by printing and copying the form as available on the World Wide Web (http://www.bgbm.fu-berlin.de/iapt/registration/regform.htm).

Registration offices are presently being arranged for in as many different countries as possible. They will serve (a) as mailboxes and forwarding agencies for registration submissions and (b) as national repositories for printed matter in which new names published locally appear.

A permanently updated address list of all functioning national registration offices is being placed on the World Wide Web (http://www.bgbm.fu-berlin.de/iapt/registration/offices.htm). This list will also be published annually in the journal Taxon.

Registration date
The date of registration, as here defined, will be the date of receipt of the registration submission at any national registration office or appropriate registration centre. For accredited journals or serials (and, for the duration of the trial phase, for publications scanned at the registration centres), it will be the date of receipt of the publication at the location of the registration centre (or national office, if so agreed).

For the duration of the trial phase, i.e. as long as registration is non-mandatory, the date of a name will, just as before, be the date of effective publication of the printed matter in which it is validated, irrespective of the date of registration. Nevertheless, the registration date will be recorded, for the following reasons:
to make clear that the name was published on or before that date, in cases when the date of effective publication is not specified in the printed matter;
to assess the time difference between the (effective or stated) date of the printed matter and that of registration, since it is envisaged that the date of registration be accepted as the date of names published on or after 1 January 2000.

It is therefore in the interest of every author to submit nomenclatural novelties for registration without any delay, and by the most rapid means available.

Access to registration data
Information on registered names will be made publicly available as soon as feasible, (a) by placing them on the World Wide Web without delay in a searchable database (http://www.bgbm.fu-berlin.de/iapt/registration/regdata.htm), (b) by publishing non-cumulative lists biannually, and (c), hopefully, by issuing cumulative updates on a CD-ROM-type, fully searchable data medium at similar intervals.

Registration as a positive step (comments of Karen L. Wilson, Royal Botanic Gardens, Mrs Macquaries Road, Sydney, N.S.W. 2000, Australia.]

Registration of nomenclatural novelties seems to me a natural way to go, heading into the 21st Century. It will enable us to find quickly what new names have been published, and to be sure that we have not missed any new name hidden in the paper mountain of botanical literature that comes out each year around the globe. This is particularly important for one-off publications (floras, field guides, etc.), which are notorious for `hiding' new names.

Some people seem to think that registration implies censorship, but this is wrong. As in the current Index kewensis all names will be listed, and without comment as to status, and as soon as received at one of the registration centres. My only caution to those looking at the mechanisms for making registration effective is that they should ensure there is a large network of registration centres or offices spread evenly around the world. This is necessary to make it easy to submit novelties for registration, given the apparently worsening state of mail services in all areas.


DATABASE ON THE TAXONOMY AND MORPHOLOGY OF THE GENUS LOTUS S.L. (LEGUMINOSAE-PAPILIONOIDEAE-LOTEAE)

T.E. Kramina and D.D. Sokoloff
Higher Plants Department
Biological Faculty,Moscow State University, 119899 Moscow, Russia
e-mail: tanya@florin.ru ; sokoloff@dds.srcc.msu.su

The taxonomy of the genus Lotus is rich in complicated problems. On the one hand, the boundaries of the genus and its subdivisions are often discussed. On the other hand, the delimitation of species and ifraspecific taxa is sometimes doubtful, as for instance in Lotus corniculatus complex, Lotus creticus complex, Lotus angustissimus complex, and in a group of perennial species of subgen. Syrmatium.

In a broad sense, the genus Lotus comprises, according to different views, from 100 species (Polhill, 1981) to 176 species (Kirkbride,1994) on all continents except the Antarctic. The majority of species occur in the Mediterranean region, Macronesia, and in the western part of North America, especially in California. Several species are cultivated as forage plants.

J.H. Kirkbride (1994) presented the worldwide Checklist of the species of Lotus s.l.

The aim of the present database is maintaining data on the taxonomy, morphology and geography of Lotus species. Taxonomic boundaries of the genus Lotus are accepted according to J.H. Kirkbride (1994), i.e. the genus is treated in the wide sense.

FLORIN Information System developed by DataX/FLORIN Inc. was chosen as a basic software for maintaining Lotus database. The System allows to store in a database large amounts of information, keep them in a good order, browse data using queries-by-example, generate configurable reports, labels and digital maps for herbarium specimens, and includes other useful possibilities.

The database is maintained by a personal FLORIN version for MS-DOS. It contains various information, such as morphological descriptions, bibliographic citations, taxonomic notes for taxa of different ranks, and also data on type material and herbarium specimens.

The taxonomic part of the database is being regularly transferred to Unix platform, and the current version of it is available in the Internet (the URL:http://www.florin.ru/florin/db/lotus.htm). Data on herbarium specimens and maps of distribution areas of species and ifraspecific taxa exist only in a personal MS-DOS version of the Database.

On the first step of database compilation nomenclatural data were included. The latest version of the database (November 1997) contains all specific names published in the genus Lotus since 1753 (507 names).The first citation of each name is available. 193 species of Lotus are accepted and attached to sections and subgenera according to our point of view. 34 names are included in database as incertae sedis. The remaining 286 names are treated as synonyms.

On the second step we plan to insert in the database the types of all taxa. At the present time, 158 types of species and ifraspecific taxa are included. Now the database also contains about 100 morphological descriptions and 350 citations of herbarium specimens (with geographical coordinates for the majority of them).

References
Kirkbride, J. H., Jr., 1994. - Taxonomic Circumscription of the Genus Lotus Linnaeus (Fabaceae, Loteae), Its Tribal Position, and Its Species, pp. 11-15, in Proc. 1 Intern. Lotus Symposium. St.Louis.
Polhill, R. M., 1981. - Loteae DC., Coronilleae (Adans.) Boiss., pp.371-375, in R. M. Polhill & P. H. Raven (eds.) Proc. Intern.Legume Conf., Kew, 24-29 July 1978. 2. Advances in Legume Systematics 1. Kew.


Current Literature

Seed pod shattering in the genus Lotus (Fabaceae): a synthesis of diverse evidence. Grant, W. F. Canadian Journal of Plant Science 76 (3): p.447-456. 1996.
Birdsfoot trefoil (Lotus corniculatus L.) is a perennial dehiscent species with an indeterminate growth habit. Pod shattering (shedding) has been a major problem, seed loss was high due to continuous flowering and time of pod maturity. The anatomy of the pod plays a role in pod shattering. A change in the orientation of pericarp cells, unequal swelling and shrinkage occurs, and a lower lignification of the mesocarp were considered major causes of shattering. The relative humidity (RH) at the time of harvest was also a major factor. The critical RH for dehiscence varied with genotypes between 35 and 49%. There was no correlation for pod dehiscence between plants grown to maturity under greenhouse conditions and the same plants grown in the field. Management practices (time of harvest, clipping early in the season, misting, mowing and turning the windrow during drying, and desiccants) were not successful in controlling pod shattering. Shattering resistance is highly heritable and is considered to be controlled by more than one gene in Lotus. Breeding to reduce shattering through recurrent selection was unsuccessful. Attempts to transfer the indehiscent seed pod trait from distantly related indehiscent species via interspecific hybridization, diploid bridge species, amphidiploidy, and backcrossing to birdsfoot trefoil, or similarly by interspecific somatic hybridization, have shown promise. The molecular approach has so far not been attempted but since genetic transformation can be carried out in Lotus, this avenue should be investigated.

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Heterospecific pollen transfer between sympatric species in a midsuccessional old-field community. McLernon, S. M.; Murphy, S. D.; Aarssen, L. W. American Journal of Botany 83 (9): p.1168-1174. 1996.
The cumulative (season-long) incidence of heterospecific pollen transfer (HPT) was examined using nine sympatric species in a midsuccessional old field in Bedford Township, Ontario, Canada. Inflorescences were collected weekly during the flowering season, and the proportion of foreign pollen/stigma was recorded. Flowering phenologies of sympatric species and ovule and seed counts of study species were also recorded. Heterospecific pollen was detected on some stigmas of each species. Medicago sativa (Fabaceae) received the most foreign pollen; in some cases, all of the grains on a stigma were heterospecific. Lotus corniculatus (Fabaceae) received the least amount of foreign pollen; the incidence of heterospecific pollen was near zero in most cases. The mean and range of foreign pollen received varied by as much as an order of magnitude between species. The six species with zygomorphic flowers, all Fabaceae, received more heterospecific pollen than the three species with actinomorphic flowers, Potentilla recta, P. simplex (Rosaceae) and Ranunculus acris (Ranunculaceae). This probably reflects a bias because the data were analysed on a cumulative basis and the Fabaceae had longer flowering phenologies. HPT was not correlated with the species' relative abundance within the community. Proportion of foreign pollen received varied temporally within species, and this variation generally was not related to phenology of any sympatric taxa or the species' own phenology. Pollen grain diameter was positively related to levels of foreign pollen received by species. This might be caused by poor adhesion of large pollen grains to small stigmatic papillae or if generalist pollinators carrying large amounts of heterospecific pollen visit the large-grained species and specialists with little foreign pollen visit the small-grained species. The large proportions of heterospecific pollen on stigmas of many species indicate that HPT occurs frequently in the community studied and the implications may include reduced seed set because of occlusion by foreign grains. As yet, however, it is unclear how important a factor HPT is in mediating pollen limitation of reproductive success.

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The origin of the Lotus corniculatus (Fabaceae) complex: a synthesis of diverse evidence. Grant, W. F.; Small, E. Canadian Journal of Botany 74 (7): p.975-989. 1996.
Although sometimes defined to have diploid populations, L. corniculatus essentially appears to be tetraploid. Biochemical and genetic evidence indicates that it is an allotetraploid. Although about a dozen diploid species have been proposed as ancestral to L. corniculatus, the evidence points strongly to four. L. uliginosus uniquely shares a rhizomatous habit, an acyanogenic factor, and a tannin characteristic with L. corniculatus, clearly showing an especially close relationship, and reflecting the possibility that it is one of two direct parental species. Principal candidates for the second parent include L. alpinus, L. japonicus and L. tenuis. The phenetic analysis presented here accords well with the hypothesis that L. corniculatus arose as a hybrid of L. tenuis and L. uliginosus. Cytogenetic evidence indicates that L. japonicus is especially closely related to L. corniculatus. Flower colour in the hybrids between L. uliginosus and L. tenuis suggest maternal inheritance and that L. tenuis could have been the female parent. An evolutionary sequence is suggested in which L. uliginosus hybridized with L. tenuis, followed by introgression from L. alpinus and L. japonicus. Alternatively, a prototype of L. alpinus, L. japonicus and L. tenuis could have been the female parent of L. corniculatus, based on evidence from maternal inheritance of flower colour intensity and interactions of Rhizobium strains.

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Some new results on the host range of Macrophomina phaseolina in Hungary. Kadlicsko, S. Acta Phytopathologica et Entomologica Hungarica 29 (1-2): p.61-66. 1994.
The importance of M. phaseolina is continually increasing in Hungary. The host range of the fungus in a small plot inoculation experiment was studied. The plant species infected by the pathogen were regarded as susceptible host plants. Among the 37 cultivated plants examined, 28 became diseased. The infected plants included crops sown over large areas (maize, sunflower, lucerne, sugarbeet and potato) as well as papilionaceous plants (e.g. lucerne, sainfoin (Onobrychis viciifolia), red clover (Trifolium pratense) and birdsfoot trefoil (Lotus corniculatus)). They were highly favourable for the propagation and survival of the fungus. The proportion of susceptible weed plants, though somewhat lower (4 out of 10 tested species) is very important because of their wide distribution.

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Growth habit of Lotus tenuis shoots and the influence of photosynthetic photon flux density, sucrose and endogenous levels of gibberellins A1 and A3. Clua, A.; Bottini, R.; Brocchi, G. N.; Bogino, J.; Luna, V.; Montaldi, E. R. Physiologia Plantarum 98 (2): p.381-388. 1996. Paper presented at the European Symposium on Photomorphogenesis in Plants, Sitges, Spain, 9-13 July 1995.
Single-node explants of L. tenuis cv. Tressur-Chaja with one axillary branch each were incubated in darkness with different concentrations of sucrose, gibberellin A3 (GA3), B9 (daminozide) or their combinations, or whole plants were treated with full sunlight, artificial shade (AS), AS + uniconazole, or AS + cimectacarb (trinexapac). A high sucrose concentration induced diagravitropic growth of the axillary branches. GA3 reversed the effect of sucrose, both in shoot sections and whole plants, inducing orthotropic growth and diminishing the sucrose levels at the shoot bases. High irradiance induced a prostrate growth of the shoots. Artificial shade increased both internode and branch length and induced an upward curvature of the shoots. This was correlated with a lower sucrose concentration and increased content of GA1 and GA3. Cimectacarb and uniconazole reversed the AS effects, although the sucrose content in the shoots remained low. These results suggest that gravitropism of L. tenuis shoots under different light intensities, but similar light quality, is controlled by the levels of GA1 and GA3 and sucrose.

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Legume seeding trials in a forested area of north-central Washington. Java, B.; Everett, R.; O'Dell, T.; Lambert, S. Tree Planters' Notes 46 (1): p.19-27. 1995.
Sowing nitrogen-fixing species is a proven silvicultural practice to increase site nutrient capital, but species' responses are site specific. Alsike clover (Trifolium hybridum), white clover (T. repens), black medic (Medicago lupulina), cicer milkvetch (Astragalus cicer), two varieties of birdsfoot trefoil (Lotus corniculatus), and Hederma pine lupine (Lupinus albicaulis), all commercially grown species, were planted at several altitudes on the Wenatchee National Forest in Washington state on recently burned disturbed forest sites. After 2 yr, alsike clover and Hederma pine lupine were the most successful species on high altitude sites (>1219 m), and black medic and Hederma pine lupine were the best performers on low altitude sites. Average total nitrogen inputs from top growth of planted species during the 1991 growing season were 20 -115 kg/ha. Nitrogen delivered by atmospheric fixation was 6-40 kg/ha. It is concluded that legumes can successfully establish in eastern Washington, ameliorating losses in nutrients after logging and residue treatment. Legumes increased total nitrogen on these sites and enhanced nutrient cycling in planted areas.

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Cytology of 2N pollen formation and pollen morphology in diploid Lotus tenuis (Fabaceae). Rim, Y.W., and Beuselinck, P.R. American Journal of Botany. 83:1057-1062. 1996.
Two genotypes of Lotus tenuis Waldst & Kit. ex Willd. PI 204882, a diploid (2n = 2x = 12), were identified as producing 2n pollen (maximum = 6%). The objectives of this research were: (1) to determine the mechanism(s) of 2n pollen formation in the L. tenuis genotypes and (2) to morphologically describe n and 2n pollen using light and scanning electron microscopy. Meiotic studies revealed that 2n pollen resulted from bipolar spindles during anaphase II of microsporogenesis. The 2n pollen germinated well, although abnormal pollen tubes were observed. The genetic constitution of 2n pollen resulting from bipolar spindles is equivalent to first division restitution (FDR) of meiosis. Fresh and air-dried pollen samples exhibited differences in size and shape under light and scanning electron microscopy. The size of diploid (2n) pollen was larger than that of haploid (1n) pollen. Normal haploid (1n) pollen was globose-prolate in shape, while diploid (2n) pollen was tetrahedral in shape.

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Use of poultry manure and plant cultivation for the reclamation of burnt soils. Vazquez, F.J.; Petrikova, V.; Villar, M.C., and Carballas, T. In: Biology and fertility of soils. 22:265-271. 1996.
Annual (Pisum sativum L. and Vicia sativa L.) and perennial (Trifolium repens L. and Lotus corniculatus L.) leguminous species were grown in pots containing samples from the ash layers of two Cambisols under Pinus sylvestris L., which has been affected by high-intensity wildfires 3 and 15 days before the sampling. The gramineous Lolium perenne L. was cultivated as a second plant after Trifolium and Lotus harvesting. Three treatments were compared: soils without fertilization and soils fertilized with two doses of poultry manure (1 and 2 g total N kg-1 dry soil). The aim of the work was to study the capacity of the ash layer to sustain vegetation and the influence of plants and organic manure on the recovery of vegetation cover, ash layer fixation and soil structure formation to avoid erosion. The ash samples were able to sustain vegetation without fertilization. The organic manure increased the yields of all the plants tested, the lower dose being the optimal for the first crop whereas the higher dose was beneficial for the second crop. The annual legumes grew very quickly. The mixture of Trifolium and Lotus seemed very suitable for reclamation of soil degraded by wildfires because Trifolium produced more phytomass than Lotus in the first growing stages whereas the development of Lotus was higher in the later growing stages. Ash layer conditions did not inhibit nodulation, which was, however, stimulated by the organic manure, particularly in the case of Lotus. Lolium after perennial legumes was the best plant combination because it produced the highest phytomass, particularly root phytomass, and thus improved vegetation cover and ash layer fixation. All the plants tested improved the formation of soil aggregates, particularly the combination of perennial legumes and Lolium. However, wet aggregate stability was higher when plants were grown on soils fertilized with poultry manure than when plants were cropped on unmanured soils, which points to the favourable influence of the organic manure on soil aggregation.

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Uptakeof phosphorus from different sources by Lotus pedunculatus and three genotypes of Trifolium repens. 1. Plant yield and phosphate efficiency. Trolove, S. N.; Hedley, M. J.; Caradus, J. R.; Mackay, A. D. Australian Journal of Soil Research. 1996. V 34:1015-1026.
The breeding of phosphate (P) efficient pastoral legumes could reduce the amount of fertilizer required on pastoral farms. Lotus pedunculatus (L. uliginosus) and 3 genotypes of white clover (Trifolium repens) known to differ in their ability to respond to added P were grown on unfertilized soil and soil to which either monocalcium phosphate (MCP) or North Carolina phosphate rock (NCPR) were added. White clover genotype 8D (a selection line from Crau) had a greater (P<0.05) internal P efficiency (shoot DM production per unit plant P) than 1A (a selection line from Gwenda) or lotus on unfertilized soil, whereas on fertilized soil, lotus had a significantly higher (P<0.05) internal P efficiency than 2 of the white clover genotypes on MCP-fertilized soil (P<0.01) and all 3 white clover genotypes on NCPR-fertilized soil (P<0.01). Lotus also had a higher (P<0.01) external P efficiency (total P uptake) than all 3 white clover genotypes on the 2 fertilized treatments. This was due to a greater root length, not a greater P uptake per unit length.

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Uptake of phosphorus from different sources by Lotus pedunculatus and three genotypes of Trifolium repens. 2. Forms of phosphate utilised and acidification of the rhizosphere. Trolove, S. N.; Hedley, M. J.; Caradus, J. R.; Mackay, A. D. Australian Journal of Soil Research. 1996. V 34:1027-1040.
To investigate differences in the ability of legumes to acidify their rhizosphere and extract various forms of P, Lotus pedunculatus (L. uliginosus) and 3 genotypes of white clover (Trifolium repens) that varied in their ability to respond to added P were grown in root study containers with either unfertilized soil, or soil fertilized with either monocalcium phosphate (MCP) or North Carolina phosphate rock (NCPR). After 6 weeks of plant growth, the containers were dismantled and 10 thin soil slices were taken 0-8 mm from the rhizoplane. These slices were fractionated for various P forms and pH was measured. All 4 legumes, especially lotus, acidified their rhizosphere. This resulted in negligible depletion of the small, H2SO4-Pi fraction (Pi, inorganic phosphorus) of unfertilized and MCP-fertilized soil, but caused direct dissolution of NCPR. The predominant forms of P depleted in the MCP-fertilized soil were the resin-Pi and NaOH-Pi forms, whereas in the NCPR treatment the H2SO4-Pi fraction was depleted. No significant depletion of any P fraction was observed in the unfertilized soils as plants were very small and P change in the soil slices was below that which could be measured using the expe