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Improving Crop Quality and Minimizing Nitrate Leaching

Through Manipulation of Ammonium Nitrate Ratios of

Fertilization Programs
The primary goal of our research is to mitigate nutrient runoff from nursery
production sites. To reach this goal, the studies in our laboratory encompass: (1) the
determination of nutrient uptake dynamics during crop production so that fertilizer can be
applied at the rates needed during the production periods when crops are actively taking
up nutrients; (2) the development and refinement of closed production systems such as
hydroponic culture, where nutrient solutions are completely recycled; (3) the
development of production methods which utilize more ammoniacal- nitrogen (NH4-N),
which does not leach as readily as nitrate-nitrogen (NO3-N); and (4) the development of
products that can be used in ornamental production which minimize nitrification (NH4
In our grant, we commissioned to look at the effects of nitrogen form (NH4, NO3) on
crop development in lilies, lisianthus and limonium, with the goal of possibility utilizing
more NH4-N than NO3-N (research programs 2 and 3). Our first attempt to grow lilies
hydroponically in winter 2002-2003 was not successful. The primary failure was that the
bulbs were submerged in nutrient solution. These bulbs rotted even though the nutrient
solutions were well aerated. We are now repeating that study this winter, with the same
closed recirculating system, but using drip irrigation with bulbs in perlite rather than
being submerged in solutions. Our research with Lisianthus and limonium will resume in
the spring after the lily crop.
During the spring and summer of 2003, a visiting scientist joined our laboratory. We
had the opportunity of having her conduct studies in the growth of floriculture crops,
through funding provided by her government. With the hydroponic system designed and
constructed from KKRF funding, she has investigated the use of the nitrification
inhibitor, N-Serve, in hydroponically grown gerberas. Gerbera was selected as the model

plant since this is a major crop in the United States and her country. The research report
of that study is presented below:

The Effect of N-Serve, a Nitrification Inhibitor, on Gerbera Growth, Flower

Production and Microbial Populations in Hydroponic Production Systems

Greenhouse growers are facing strict governmental regulations concerning the
discharge of spent nutrient solution in order to abate ground water pollution resulting
from nutrient salts and agricultural chemicals. At the same time the use of recaptured
irrigation water by the agricultural/horticultural industry is increasing due to increased
pressure by water purveyors and governmental agencies to reduce water usage. When
used as a completely closed recirculating system, the above would seem to be a solution
to the problem of pollution and water conservation.
Although the use of recycled irrigation water will reduce water usage and ground water
pollution, serious grower concerns exist regarding:
spread of phytopathogenic (Wohanka, 1991) and deleterious microorganisms
(Van peer and Schippers 1989) in the recycled water, and
the accumulation of inorganic (Jensen and Collins, 1985) and organic
compounds (Yu and Matsui, 1993) in the recycled water that may cause
yield-reducing damage to the plants .
With respect to pathogen management, several methods for disinfesting the nutrient
solution have been developed for the use in closed hydroponics: heat treatment, ozone,
UV radiation, and slow sand filtration. These methods are known to reduce the
pathogenic microflora but may also reduce the beneficial microflora, which may play a
role in suppressing diseases (McPherson et al., 1995; Postma, 1996, Postma et al., 1999
and 2000, Tu et al., 1999). For example, Pythium can disperse very rapidly in a sterilized
environment, while its growth in a non-sterilized environment is much slower.
Similar phenomena can be seen with certain Phytophthora species.
The pressure to use closed growing systems has also stimulated research on the
presence of metabolites produced by plant roots and/or microorganisms in the

recirculating solution (Waechter- Kristensen et al., 1994 and 1999). It has been reported
that certain metabolites (carboxylic acids, sugars, amino acids) may be beneficial to plant
growth at low concentrations, whereas growth reducing effect may occur at higher
concentrations (Jensn & Adalsteinsson 1993).
In this context, the first objective of the our study was to evaluate the in vitro effect of
N-Serve on nitrifying bacteria and on zoospores of Phytophthora cryptogea.. Second, we
evaluated the in vivo effects of N-Serve on the culturable aerobic heterotrophic bacteria
(AHB) and its stabilising effect on the pH and electrical conductivity (EC) of the
hydroponic nutrient solution used in the cultivation of gerbera. Additionally, plant growth
was evaluated: flower production, stem length and the plant dry weight.
Material and Methods
A. N-Serve (a.i. Nitrapyrin): N-Serve is an ammoniacal nitrogen stabilizer which inhibits
the nitrification of ammoniacal and urea nitrogen fertilizer in the soil by selectively and
temporarily inhibiting Nitrosomonas species. These bacteria convert ammonium
nitrogen to nitrite nitrogen, which in turn is converted to nitrate nitrogen by other
bacteria, e.g. Nitrobacter.
N-Serve 24E consists of 22.2% 2-chloro-6-trichloromethylpyridine, 2.5% related
chlorinated pyridines including 4,6-dichloro-2-trichloromethylpyridine, and 75.7%
Xylene-range aromatic solvent. As reported on the literature, Nitrapyrin has a very
selective effect on Nitrosomonas bacteria and is reportedly bacteriostatic or bactericidal.
B. Laboratory studies: Several in vitro studies were conducted to determine the NServe concentration required to inhibit zoospore germination of Phytophthora cryptogea
and its effects on the aerobic heterotrophic bacteria populations after application to a
nutrient solution. Samples of gerbera nutrient solution from the greenhouse experiment
were treated at 0, 25, 50 and 100 ppm: after 30 minutes bacteria populations were
measured by plating a dilution series onto Tryptic soy agar substrate and incubated at 25
C for two days.

Hydroponic studies. Gerbera plants were grown in a closed recirculating hydroponics

system (Figures 1 and 2). Nutrient solutions contained 16 mg NH4NO3, 482.23 mg
KNO3, 708.45 mg Ca(NO3)2*4H2O, 52.82 mg (NH4)2HPO4, 81.6 mg KH2PO4, 1.73 mg
H3BO3, 283.45 mg MgSO4*7H2O, 0.049 mg CuSO4*5H2O, 3.48 mg K2SO4, 1.72 mg
ZnSO4*H2O, 0.61 (NH4)6Mo7O24*4H2O, 0.79 mg MnCl2, 28.93 mg Fe EDTA (98%) per
liter water, and the pH was adjusted with H3PO4 and KOH.
There were two treatments: (1) N-Serve application at 25 ppm, and (2) no N-Serve
application. Plants were planted in black polyethylene citrus pots filled with #2 perlite,
and then placed in a recirculating hydroponics system (Figures 1 and 2). During the
three-month growth period, plants were monitored for flower production and nutrient
solutions were monitored for pH, electrical conductivity, and temperature. At the end of
the production cycle plants were harvested, dried and weighed.
Additionally, we monitored the effect of N-Serve on the AHB populations in amended
and unamended systems. Nutrient solution from each treatment were sampled every week
and the bacteria populations were estimated by plating a dilution series onto Tryptic soy
agar substrate and incubated at 25 C for two days.
Results and Discussion
In Vitro Studies: Preliminary in vitro tests indicated that the N-Serve concentration
required to inhibit zoospores germination of the P. crytogea in nutrient solution is
approximately 25 ppm (Figure 3). In addition, we discovered that within 30 minutes
following the application of 25 ppm N-serve to the nutrient solution, there was about
40% reduction in the aerobic heterotrophic bacteria, and about 90% reduction at 50 and
100 ppm (Figure 4).
Greenhouse Studies: Bacteria populations

Based on the data from the above

laboratory tests, we tested N-Serve in the hydroponic system at 25 ppm. However after
the initial reduction in population, the general bacteria population increased significantly
within 3 day (Figures 5 and 6).

Additionally, our preliminary study indicated that the bacterial species diversity also
changed following application of N-Serve. Specifically there was a significant increase
of fluorescent pseudomonads in the N-Serve treatment compare to the control.
Greenhouse Studies Nutrient Solutions. The pH of nutrient solutions without NServe (treatments 2 and 3) decreased during the study (Figure 7). Therefore, the pH of
these solutions had to be increased to pH 6.0 with the addition of potassium hydroxide.
Nutrient solutions supplemented with N-Serve were stable throughout the period of the
study (Figure 7). The electrical conductivity of nutrient solutions gradually increased
from 1.6 mS/cm to 1.8 to 2.0, depending on the treatment (Figure 8). Temperature of
nutrient solutions gradually decreased from 25oC to 15 oC during the course of the study
(Figure 9).
Greenhouse Studies Plant Development Plant growth was variable between
experiments. In experiment A, plants treated with N-Serve had significantly larger leaf
dry weight than the controls (Figure 10). However, in experiment B, there were no
differences in shoot dry weight between treatments (Figure 11). Root dry weights did not
differ between treatments for experiment A (Figure 12) and experiment B (Figure 13).
Total number of flowers produced were significantly different for plants treated with NServe versus those plants not treated with N-Serve in experiment A but not in experiment
B (Figure 14). The average stem length did not differ between treatments in experiment
A and experiment B (Figure 15).
Future studies.

Based on the above results, additional investigations are being

conducted in Dr. Stanghellinis laboratory to elucidate the efficacy of N-Serve as a

fungicide and as a chemical which may enhance, after an initial reduction, bacterial
populations in the recycled nutrient solution and its positive effect on plant growth and
flower production.