PROPUSE OF SEAWATER IRRIGATION CROPS

Salicornia bigelovii as an Example With the Association of Nitrogen Fixers Bacterium (Plant Growth Promoting Bacterium- PGPB)

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Instructor:

Dr. Edgar Omar Rueda Puente Sonora university Member of the National System of Researchers Sonora, México Tel phone Of. + 01-6625960297 in Agriculture Departament of Sonora University. [email protected] [email protected] [email protected]

1 Picture obtained from: seaphire internacional.

OUR JUSTIFICATION WITH HALOPHYTES:

WHY WE ARE WORKING WITH Salicornia bigelovii

Development of Halophytic Crops

Availability of fresh water for agricultural uses continues to decline due to increased demands for urban uses along with shrinking groundwater and surface water reserves. When this is coupled with predicted reductions in rainfall and streamflow that may occur as a consequence of the "greenhouse effect" that will impact major irrigated areas such as Arizona, it is clear that the need for salt tolerant crops will become even greater than at present. In addition to increasing the traditional breeding efforts presently underway to increase salt tolerance within specific crop species, that need should be addressed through the use of as many additional approaches as possible. For example, advances in biotechnology that are making it feasible to consider transfer of genetic information from almost any source into crop plants suggests that large increases in salt tolerance of crops may realistically be expected in the near future. The major constraint to accomplishing that feat is identifying the characteristics and or mechanisms to be transferred into the crop plants. In addition to being potential sources of the required genetic information for the desirable characteristics, halophytes also are ideal experimental model plants to use in the attempt to identify those mechanisms and characteristics.

The typical approach to studying salt tolerance is to compare plants (both sensitive and tolerant plants) subjected to excess salinity with plants not subjected to salinity, looking for responses to the added salt. Examples of such responses are production of unique proteins or large amounts of presumed compatible osmotic solutes such as proline and glycinebetaine. The difficulty with such an approach is that it is difficult to distinguish those responses that are truly adaptive from those that are reflections of metabolic lesions.

There is an alternative approach that is possible only with extreme halophytes, i.e., those plants are highly adapted to growing in environments with persistently high salinity. In those plants, the adaptation to high salinity has involved giving up the ability to grow as well as at low salinities that still are high enough to inhibit growth of glycophytes. That is, the optimum salinity for growth has shifted from zero in glycophytes and many less tolerant-halophytes to 75-150 mM in many of these highly tolerant halophytes, with decreased growth observed at both higher and lower salinities. Thus, if one compares the responses of those plants to LESS THAN optimum salinity as well as greater than optimum levels with plants grown at optimum salinity, it should be possible to sort out those responses that are truly adaptive from those that are the result of lesions or other forms of damage. A mechanism or process, e.g., that has adapted to now function better at substantial levels of salinity might be expected to function less well at relatively low levels of salinity and result in less growth at those levels of salinity. The challenge is to identify those mechanisms. In addition to contributing a better understanding of salt tolerance per se, this also should be a productive way of identifying those traits that could be transferred to crop plants and contribute to increased salt tolerance.

PREVIOUS WORK AND PRESENT OUTLOOK

There are two ways in which halophytes can contribute to development of halophytic crops. The first involves domesticating the halophyte by introduction and/or genetic improvement of crop characteristics. The second involves identifying the characteristics, processes, mechanisms, etc. that are responsible for the high salt tolerance in the halophyte and transferring those characteristics to present crop plants.

The first approach has been focusing on the considerable attention during the past decade. The potentia1 uses of halophytes as irrigated crop plants have been reviewed by O'Leary (1984), and research during the past decade has validated the concept of obtaining high productivity from halophytes when irrigated with highly saline water (O'Leary et al., 1985; Pasternak et al., 1985; O'Leary, 1988). Most of the emphasis has been on identifying and selecting halophytes that might be valuable as forage/fodder crops (O'Leary, 1986; Watson et al., 1987) however; a potential oilseed crop whose vegetable oil is high in unsaturated fatty acids also has been identified (Glenn et al., 1991). Thus, it is conceivable that, with genetic improvement of the crop characteristics of these highly productive halophytes, some useful halophytic crops can be developed.

There has been a reasonable amount of information published on the physiology of halophytes in the past. A considerable amount of attention has be en devoted to documentation and analysis of growth reductions due to excess salinity in halophytes as well as glycophytes, including crop plants. However, no attention has been given to determining the causes for the growth reduction at low salinity in those halophytes that have growth optima at salinity levels such as 75-150 mM.. In the few instances where anyone has speculated about the cause of the reduced growth at less than optimum salinity, it is attributed to lack of sufficient solutes in the leaves to generate turgor (Jennings, 1976; Flowers et al.,1977; Munns et al.,1983; Flowers and Yeo, 1986), even though that hypothesis is not well supported with data.

Since growth depends on substrate availability as well as sufficient turgor, it is reasonable to question the effect of the less than optimum salinity on photosynthesis. The effect of salinity on photosynthesis in halophytes has been investigated, and in spite of the fact that the focus usually was on comparing optimum versus excess salinity, in some cases data were obtained for a range of salinity levels that enable one to compare photosynthesis rates at less than optimum salinities with those at optimum levels. In Salicornia, arguably the most salt tolerant C3 vascular plant, photosynthesis was higher at -32 bars osmotic potential (Tiku, 1976) or 342 mM salinity (AbdulRahman and Williams, 1981) when measurements were made at several salinity levels, however, Pearcy and Ustin (1984) found no differences from O to 450 mM. In Atriplex nummularia, photosynthesis was higher at leaf water potentials of -1.5 to -2.0 MPa than at either higher or lower water potentials (Pham Thi, 1982). In Leptochloa fusca, grown in the absence of NaCl or with NaCl at 250 mM, photosynthesis was higher in the presence of added NaCl than when it was absent at 32°C or 39°C, but the reverse was true when the temperature was 19° C (Gorham, 1987). In all cases there was insufficient information to determine whether the lower photosynthetic rates at the lower salinities (or higher water potentials) were due to stomatal or non-stomatal effects.

In fact, even in the cases where investigators have demonstrated the reduced photosynthetic rates at excessive salinity levels in halophytes, the picture is unclear. Some have attributed the reduced photosynthesis to reduced leaf conductance (Farquhar et al., 1982; Guy et al., 1986a, 1986b) while others have concluded that photosynthesis was reduced independent1y of changes in stomatal conductance (Ball & Farquhar, 1984; Longstreth et al., 1984; Pearcy & Ustin, 1984). Furthermore, in some cases it has been concluded that growth was reduced by some factors other than photosynthesis, and the net effect was due to reduced photosynthetic surface rather than reduced photosynthetic rate per unit leaf surface (De Jong, 1978; Gale & Poljakoff-Mayber, 1970; Winter, 1979).

Some of the differences may be due to real differences among species, but some are also the result of differences in experimental conditions and differences in the manner in which the data are expressed. It depends on whether the photosynthetic rate is expresses on a leaf area, leaf weight, or chlorophyll basis. Depending on which is used, the photosynthetic rate may be shown to be longer or higher (Winter, 1979). Salicornia bigelovii as an Example With the Association of Nitrogen Fixers Bacterium (Plant Growth Promoting Bacterium- PGPB) I. Introduction

One of the scarcest and most finite resources in the world is fresh water. Specifically, fresh-water for agriculture. On this planet, only one half of one percent of the water is fresh water, the other 99.5 % being locked up on polar ice caps or the sea. The sea covers 2/3 of the Earth’s surface. The remaining 1/3 of the Earth’s surface is land, with 1/3 of that being desert. That desert has 22.000 miles of coastline.

To the Agricultural laboratory “Dr. Félix Ayala Chairez” into the Sonora University, this presents an essentially untapped resource for the benefit of the environment and the Earth’s people -the challenge of providing food crops grown on seawater, and realizing research and experimentation with halophytes -salt tolerant plants – to developed the technologies to contribute to the world’s food supply, make the deserts bloom and be beneficial to the planet as a whole. This critical research and development must continue into the next century.

II. Social, ecological and economic potential

The cultivation of Halophyte crops, Salicornia bigelovii in particular, has utilized marginal and which has little or no value for conventional agricultural crops. Its natural habitat is sandy soil close to the ocean -where the only irrigation source is seawater. There are genotypes, the first Halophyte selected for agricultural development has been grown and harvested on demonstration plots and farms in México, Egypt, The United Arab Emirates, Kuwait and Saudi Arabia.

The advantages of halophyte crops are the follows: a) Strong support to the agribusiness economy - with high impact on regional development b) The reclamation of crop land. Land which had been wasted and unused will be reclaimed -allowing for new coastal desert communities, more jobs and a benefit to the environment. c) The production of forage and oil seed on a large scale profitable basis. d) The production of other crops on the areas now using fresh water for crops producing forage and oil seed can be accomplished. e) The commercial production of halophyte crops, with seawater, can contribute to balancing the Earth’s CO2 cycle thus having a positive impact on the “Greenhouse effect”

III. Crop Management / Salicornia as an Example a) Planting seasons The planting season for northwest Mexico is the 15th February to 15th march. In the Middle East the recommended planting date is October lst through 30th. The growing cycle is approximately seven (7-9) months. Therefore crops are harvested in September in Mexico and in June in the Middle East.

b) Land preparation Sandy soils require disking two to three times along with the use of a cultipacker if the soil contains clay ripping may be necessary. c) Planting Methods Seeds are planted at a rate of 30 to 40 kilograms per hectare. In areas of less than one hectare hand seeders are used. Hand seeders are easily available and inexpensive. For larger plots, commercial seeders are used. Due to the small size of the Salicornia seed it is recommended that the Brillion seeder be used. The Brillion seeder contains cultipackers in front and back and a seed feeder in between, allowing the seed to be placed at a precise depth, therefore, improving germination. d) Fertilization Prior to planting, it is suggested that fertilizer be applied at the rates of 50 kilograms of NH3 and 50 kilograms of P205 per hectare. Phosphorus composition is not critical; however, the nitrogen source is very important and should be either urea or ammonia (as a gas).

From the end of the first month through the fifth month of the growing cycle, NH3 should be applied every 5 to 10 days a total of 200 to 300 kilograms per hectare needs to be added during this period. e) Cultural practices The use of herbicides and pesticides are not necessary during the growth of Salicornia to date, both seeds have not been found to be highly susceptible to many diseases, especially with reasonable management practices. f) Irrigation

Irrigation will depend upon the infiltration rate of the soil. As a general guide the soil will normally be irrigated on a schedule ranging from daily to once every 5 days. Total water required will be approximately two times the local pan evaporation rate.

g) Harvest Harvesting preparations are made by spraying desiccant (herbicides) to promote the drying of ecotypes of Salicornia. Both genotypes are then harvested using conventional combines such as those used on wheat.

If it is not possible to spray with desiccants, a windrower is used. At its physiological maturity, ecotypes of Salicornia are cut by the windrower and laid into rows to dry by the sun. After drying a thresher with a small pick-up head is used to pickup the crop from the soil. The seed is threshed and the straw is collected as forage for animals. For areas of less than one hectare, the combine is not used. Instead the crop can be threshed in a stationary thresher or, by hand.

IV. Salicornia genotypes. Results in Mexico.

In the past five years of commercial development of the crop in Baja California Sur, Kino Bay and Santa Ana, Sonora. Mexico, the yields of oil seed and forage are represented in the Table 1, as follows:

Table 1. Yield of oil seed and forage of Salicornia bigelovii. YIELD OIL SEED FORAGE (dry matter) (Kg/ ha) (Kg/ha) Minimum 500 14,000 Medium 1,500 18,500 Maximum 2,500 22,000 Soybean in Sonora and Baja California, Mexico have a minimum oil seed yield of 1,000 kg/ha and maximum of 2,500 kg/ha with the average of 2,000 kg/ha. Soybean uses fresh water and Salicornia uses seawater.

V. Infrastructure cost

According to a study produced by FIRA -bank of Mexico and Genesis (a private company dedicated to transferring this Technology to Mexico) according Mota (1990), the cost of infrastructure, based upon a one hundred hectare farm is $2,576 per hectare. This cost is approximately 20% less than a comparable infrastructure cost for a typical irrigation district using a dammed reservoir which costs $3.000 per hectare.

Table 2 exhibits total cost, per item. From this table it is clear there are two alternatives: ecotypes as a sole crop associated with aquaculture, i.e. shrimp, finfish or algae farms using sea or brackish water. When operating with the latter alternative the investment declines from $2,576 per hectare to $1,074.18 per hectare. This assumes that the entire seawater well system and other items are charged to the aquaculture activity.

Table 2. Costs of infrastructure/per hectare for a Salicornia Farm Ecotypes Salicornia Ecotype Salicornia FARM CONCEPT FARM ONLY INTEGRATED Land preparation 298.00 298.00 Cleaning 10.00 10.00 Topographical study 25.00 25.00 Leveling 263.00 263.00 Wells and Equipment 468.68 00.00 Movement of equipment 17.24 Drilling 42.80 Testing 22.08 Casing 29.84 Pump 81.70 Electric motor 96.90 Starter 68.42 Transformer 41.40 Electric lines 96.90 Irrigation Infrastructure 1,581.31 776.18 Concrete pipe 805.13 PVC pipe 776.18 776.18 Complementary 200.00 00.00 Installations House for supervisor 100.00 Share and Storage 100.00 TOTAL 2,575.99 1,074.18

NOTE: The costs are based on Kino Bay, Sonora, Mexico farm can change at other sites. Consider the continue dollar change.

VI. Production cost From the afore mentioned study produced by FIRA -bank of Mexico, production costs for salicornia is $878.30 per hectare. Fifty (50%) percent of that cost is seawater pumping expense.

Presently, Mexico is utilizing wells with PVC casing and electric or diesel pumps with 10” discharge. The cost of pumping can be reduced when more efficient systems can be implemented, such as constructing open sea canals and upgrading the 10” discharge pump to 30". The 30" pumping system is used in the salt plants located in Guerrero Negro, B.C.S. Mexico and are effective.

To make the production of Salicornia attractive to farmers, the infrastructure, including irrigation can be funded by local governments. The farmer then pays for only the seawater he uses. This system is presently, effective in Mexico

Pumping and fertilization costs can be reduce further if the Salicornia farm is integrated, with aquaculture. This assumes that the SOS-7 farm receives discharge water from the aquaculture facility at essentially zero pumping costs. Table 3 exhibits that the cost of $878.30 per hectare can be reduced to $354.71 per hectare, because was eliminated pumping and fertilizer costs. Table 3. Production Costs of Salicornia /per hectare based upon 100 hectare farm. Concept Salicornia alone Salicornia associated (dollars per ha) with aquaculture (dollars per ha) Soil preparation 82.30 82.30 Fertilization 78.44 00.00 Planting 52.91 52.91 Irrigation 535.05 89.90 Pests control 00.00 00.00 Materials (pesticides/herbicides) 00.00 00.00 Harvesting 95.27 95.27 Other 34.33 34.33 TOTAL 872.30 354.71

VI. Integrated Seawater Based Agriculture and Aquaculture

In the 1979´s The Environmental Research Laboratory, begun research in controlled environment on shore aquaculture specifically, shrimp production. This expertise is now available in the design and development of bloodstock, hatchery/nursery and production systems and feed and pathology programs.

As a result of growing shrimp on seawater, there is a by-product opportunity, that seawater with a rich nutrient source could be used to irrigate plants-halophytes.

From a base of operations in Puerto Peñasco, Sonora, Mexico, the first integrated model of this aquaculture and agriculture concept is now being developed. This prototype includes a shrimp hatchery with a total capacity of 3,000,000 post larvae per month; a grow-out of shrimp in nine raceways of 200 square meter each; a fish grow-out (Tilapia) in five raceways with an algae culture (Gracilaria algae for industrial purposes; and, a fifty hectare farm with Salicornia. The budget for this is approximately $1 million.

Currently, it is planned to have both the hatchery and the grow-out shrimp farm in operation.

Currently, we are testing the water from aquaculture rich in nutrient.

VII. Summary

This desert coast vision coupled with our aquaculture and agriculture technologies provide the basis for economically viable seawater based communities along the world´s desert seacoasts.

It is in the context that we envision the cultivation of Salicornia and Shrimp, together with Tilapia, algae and ornamental halophytes, opening up additional avenues for alternative solutions to the problem of food production…. creating new hope for the world populations.

VIII. Nitrogen fixer’s bacterium as a Plant Growth Promoting Bacterium on Halophytes: an alternative to chemicals fertilizers.

Considering the model of interaction Plant-Azospirillum bacteria, Azospirilla are free-living plant growth-promoting bacteria (PGPB) capable of affecting the growth and yield of numerous plant species, many of agronomic significance. The leading theory on its growth promotion capacity lay in its ability to produce various phytohormones that improve root growth and absorption of water and minerals that yield larger, and in many cases, more productive plants. Since its rediscovery in the mid-1970´s by the late J. Döbereiner, it has consistently proved to be a very promising PGPB. Substantial advances in exploring the genetic basis of the beneficial effects of Azospirillum and other PGPBs on plants have been made. From the extensive genetic, biochemical, and applied studies, Azospirillum is considered one of the best-studied PGPB.

Our last and another comprehensive reviews of the agricultural, environmental, and physiological aspects of Azospirillum interactions with plants, promote in our Agricultural Laboratory, enlarge the knowledge of this type microorganisms to isolate from halophytes like as Salicornia bigelovii.

Among results obtained are: that one bacterium in association to the rizhosphere to Salicornia bigelovii was identified as Klebsiella pneumoniae. Effects of K. pneumoniae under laboratory, greenhouse and field, were evaluated at the germination and early seedling growth stages of two genotypes of S. bigelovii (‘wild genotype’ and cv. ‘SOS- 10’). This bacterium, in conjunction with Azospirillum halopraeferens, was tested for growth-promoting ability when inoculated on S. bigelovii genotypes under several saline concentration conditions. During germination and early seedling growth, K. pneumoniae showed high specificity for the wild S. bigelovii genotype, while A. halopraeferens was more specific for the SOS-10 S. bigelovii genotype.

On the other hand, the growth and development of the same two genotypes of S. bigelovii, were evaluated under field conditions. When inoculated with previously selected and cultivated native strains of Klebsiella pneumoniae and Azospirillum halopraeferens, these inoculants increased some growth and development parameters, using measures of weight, length of plants, and biochemical characteristics, including total protein, ash, and total lipid content in selected plant parts. The findings suggest that yields of both genotypes of S. bigelovii, under field conditions, can be improved by the application of K. pneumoniae or A. halopraeferens strains.

This is the first report of Klebsiella pneumoniae as nitrogen-fixing bacterium associated to the halophyte Salicornia bigelovii.

VIII: References

Ayala, F.. and J. O’Leary. 1995. Growth and physiology of Salicornia bigelovii Torr. at suboptimal salinity. International Journal of Plant Sciences, 156:197-205. Bashan, Y. and Holguin, G. 1997 (a). Azospirillum-plant relationship: enviromental and physiological advances (1990-1996). Journal Microbiology, 43: 103-121. Bashan, Y. and Holguin G. 1997 (b). Proposal for the division of plant growth-promoting rhizobacteria into two classifications: biocontrol-PGPB (Plant Growth- Promoting Bacteria) and PGPB. Soil Biology & Biochemistry, 30: 1225-1228. Bashan, Y., Holguin, G., and Ferrera, C. 1996. Interactions between plants and beneficial Glenn, E., Brown J., and O’Leary J. 1998. Irrigating crops with seawater. http://www.scram..com./1998/0898issue/0898glenn.html. Science. American, 279: 76- 81. Glenn, E., Hicks, N. and Riley, J. 1995. Seawater irrigation of halophytes for animal feed. Enviromental Research Laboratory, Tucson Arizona, p 221-234 Glenn, E., Lewis T. and Moore D. 1994. Synthesis of selected reseach results on Salicornia bigelovii. Halophyte Enterprises, Inc., 60 p. Glenn, E. and O´Leary, W. 1984. Relationship between salt accomulation and water content of dicotyledonous halophytes. Plant Cell and Enviroment, 7:253-261 Glenn, E., O’Leary J. and Corolyn, W. 1991. Salicornia bigelovii Torr.: an oilseed halophyte for seawater irrigation. Science, 251:1065-1067. Mota, U. 1980. Las halófitas en el siglo XXI. En: Primera reunión nacional sobre ecología, manejo y domesticación de las plantas útiles del desierto. Instituto Nacional de Investigaciones Forestales. Monterrey, México, p. 495-500. Mota, U. 1990. Seawater irrigation crops Salicornia (SOS7) as an example. The University of Arizona ,Tucson Arizona, 12 p. Mota, U. 1996. Levels of fertilizer in Salicornia bigelovii. In: First International Technical Seminar for Salicornia. Proceedings. Planetary Design Corporation. Puerto Peñasco, Sonora, Mexico,96 p. Mota, U. 1999. Sistemas de riego aplicados a la producción de alimentos con agua de mar. I simposium internacional sobre financiamiento para modernización de áreas de riego. Hermosillo, Sonora, México, p 5. Rueda-Puente, T. Castellanos, E. Troyo-Diéguez, J. L. Díaz de León-Álvarez, and B. Murillo-Amador. Effects of a nitrogen-fixing indigenous bacterium (Klebsiella pneumoniae) on the growth and development of the halophyte Salicornia bigelovii as a new crop for saline environments. Journal Agronomy of Crops Sciences. Rueda-Puente, Thelma Castellanos C., Enrique Troyo-Diéguez, and Jose Luis Díaz de León-Álvarez. Effect of Klebsiella pneumoniae and Azospirillum halopraeferens on the growth and development of two Salicornia bigelovii genotypes. Australian Journal of Experimental Agriculture.