Aerial transmission and strategies for control of Pythium on hydroponically grown cucumbers.
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Aerial transmission and strategies for control of Pythium on hydroponically-grown cucumbers
Goldberg, Natalie Pauline, Ph.D. The University of Arizona, 1990
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AERIAL TRANSMISSION AND STRATEGIES FOR CONTROL OF PITH/UM
ON HYDROPONICALLY-GROWN CUCUMBERS
by
Natalie Pauline Goldberg
A Dissertation Submitted to the Faculty of the
DEPARTMENT OF PLANT PATHOLOGY
In Partial Fulfillment of the Requirements For the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
THE UNIVERSITY OF ARIZONA
1990 THE UNIVERSITY OF ARIZONA 2 GRADUATE COLLEGE
As members of the Final Examination Committee, we certify that we have read
the dissertation prepared by ___N_a_t_a_l_i_e __P_a_u_l_i_n_e __G_o_l_d_b_e_r~g ______
entitled Aerial Transmission and Strategies for Control of Pythium on Hydroponically-Grown Cucumbers------
and recommend that it be accepted as fUlfilling the dissertation requirement
for the Degree of Doctor of Philosophy
Date
Date
Date
Date
Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College.
I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.
/'1';.. I ffo 3
STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgement the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.
SIGNED: d~f?C&J~ 4
This dissertation is dedicated to family and friends whose love, friendship, support and encouragement has helped me to achieve my goals. 5 ACKNOWLEDGEMENTS
I wish to express my sincere appreciation and gratitude to Dr. Michael Stanghellini under whose guidance this research was conducted. I express thanks, not only for his scientific help and critical review of this manuscript, but also for his genuine personal interest, his excellent advice, his endless and contagious enthusiasm, and most of all for making my graduate research fun. I would also like to express my gratitude to Dr. Richard Hine, for his generosity, interest, friendship and for teaching me what Plant Pathology is really all about. Thanks are expressed to Dr. Robert Gilbertson, whose friendship was never ending and who always provided a great game of racquetball. And to Dr. Iraj Misaghi for continued support and sincere friendship. The kindness and generosity of Mrs. Pat Hine is also gratefully acknowledged and appreciated. Thanks also to Mr. Tom Kruk, whose sense of humor helped to bring pleasure to life without windows. Special thanks are given to Dr. Judy Brown and her family, Gary, Kevin and Katie, who are truly very precious friends. Thanks for everything, but most of all, thanks for picking me up when I was down and for readjusting my thinking whenever I was sure it wasn't worth the pain. The completion of this work would not have been possible without the help of my good friend and labmate, Mr. Scott Rasmussen. Thanks are extended for helping me with the hydroponic tanks and for always being willing to lend a hand whenever help was needed. Heartfelt thanks are extended to my many good friends I have made during my stay in Arizona. I would especially like to thank the members of Bad News, The Harvey Mason Fan Club and Triple Bypass, for providing good times and a lot of laughs. And to Terri Gerber, for helpful advice and encouragement; and to Jody Schneider, for her friendship and for giving me the opportunity to coach volleyball at Mountain View High School, a wonderful diversion without which this work would not have been possible. Finally, my appreciation is given to the University of Arizona and the Department of Plant Pathology for providing the resources necessary for me to carry out this research. 6 TABLE OF CONTENTS
Page
LIST OF ILLUS1RATIONS ...... 8
LIST OF TABLES ...... 9
ABS1RACT ...... 10
CHAPTERS
1. INTRODUCTION AND LITERATURE REVIEW ...... 12
History and definitions ...... 12 Types of hydroponic systems ...... 13 Diseases in hydroponically-grown vegetables ...... 15 Disease symptoms ...... 20 Introduction and spread of pathogens ...... 22 Methods of disease control ...... 25 Objectives ...... 30
2. FIL1RATION AS A S1RATEGY FOR CONTROL OF PITHIUM APHANIDERMATUM IN RECIRCULATING HYDROPONICS ...... 31
Materials and Methods ...... 32 Results and Discussion ...... 36
3. INGESTION-EGESTION AND AERIAL TRANSMISSION OF PITHIUM APHANIDERMATUM BY SHORE FilES (EPHYDRINAE: SCATELLA STAGNALIS) ...... 42
Materials and Methods ...... 44 Pathogen infestation of adult flies, pupae and larvae ...... 44 Germinability of oospores recovered from larvae, pupae, and adult flies ...... 46 Germinability of oospores in insect frass ...... 46 Larval transmission of P. aphanidennatum ...... 47 Adult fly transmission of P. aphanidennatum ...... 35 7 Page
Results ...... 48 Pathogen infestation of adult flies, pupae, and larvae ...... 48 Germinability of oospores recovered from larvae, pupae, and adult flies .. ,...... 48 Germinability of oospores in frass ...... 51 Larval and adult fly transmission of P. aphanidennatum ...... 51
Discussion ...... 52
REFERENCES CITED ...... 54 8 liST OF ILLUSTRATIONS
Figure Page
1. Recirculating hydroponic system ...... 33
2. Photograph of the 7-J.lITl filter cartridge used to study the efficacy of filtration of zoospore infested water on control of root rot of cucumber caused by Pythium aphanidennaP.jm ...... 35
3. Disease incidence in cucumbers resulting from the recirculation of water from (A) tank 1 which was artificially infested with Pythium aphanidermatum to (B) tank 2 and (C) tank 3 five days after receiving infested water passed through a 20-J,lm and 7-J.lITl filter, respectively ...... 37
4. Efficacy of filtration of infested water to control root rot of cucumber caused by Pythium aphanidermatum in a recirculating hydroponic system ...... 38
5. Various life stages of a shore fly (Scatella stagnalis) ...... 43
6. Photomicrograph of oospores of Pythium aphanidermatum in the cortex of a naturally infected cucumber root ...... 45
7. Photomicrograph of oospores of Pythium aphanidermatum in the gut of a shore fly larva ...... 50
8. Photomicrograph of oospores of Pythium aphanidermatum from adult fly frass ...... 52 9 LIST OF TABLES
Table Page
1. Root-infecting fungi isolated from roots of vegetables grown in recirculating hydroponics ...... 18
2. Root-infecting fungi reported as pathogens of vegetables grown in recirculating hydroponics ...... 19
3. Recovery of Pythium aphanidennatum from the 7-j.JITl filter cartridge ..... ~ ...... 40
4. Percent of adult flies, pupae and larvae infested with Pythium aphanidennatum ...... 49 10 ABSTRACT
Recirculating hydroponic cultural systems facilitate continuous dissemination of introduced plant pathogens. Chemicals are unavailable for the control of diseases in hydroponically-grown vegetables, thus alternate methods of control need to be investigated. An experiment was conducted to test the efficacy of filtration of infested water for the control of root rot of cucumber caused by Pytlzium aplzanidennatum. Germinated cucumber seedlings were transplanted into separate hydroponic tanks. Each tank received water from a common source tank which was
(a) recirculated through a 20-J,lm filter or (b) through a 7-J.lm filter. Pytlzium aplzanidennatum was introduced into the source tank on two infected plants.
Recirculation of infested water was conducted every other day for 5 days at a flow rate of 114 L/min for 30 min. One day after recirculation, 67% of the plants in the tank receiving water passed only through the 20-J.lID filter were infected and all plants were infected within 3-days. None of the plants in the tank which received water passed through the 7-J.lm filter were infected until5-days post-recirculation. A repeat experiment yielded similar results, however there was a delay in the onset of disease expression. The fungus was recovered from the surface (0 mm) and middle (8 mm depth), but not from the inner core (16 mm depth) of the 7-J.lID filter. It was cOricluded that the 7-J.lm filter was effective in removing zoospores from infested water. Since plants in the tank receiving water passed through the 7-J.lm filter eventually became infected, an alternate source of introduction and spread of the pathogen was investigated. Aerial transmission of Pytlzium aplzanidennatum by shore 11 flies (Scatella stagnalis) was documented for the first time. Shore flies which were thought to feed only on blue-green algae and diatoms, also fed on rucumber roots colonized by the fungus. Ninety-seven percent of the first and second instar larvae,
20% of the pupae/third instar larvae, and 10% of adult flies carried mature oospores in their gut. Oospores excreted by larvae and adults were germinable. Pytlzium aplzanidennatum was transmitted to healthy cucumber plants by naturally infested larvae and adult flies. Adult flies infested with P. aplzanidennatum may account for pathogen introduction and spread within commercial greenhouse facilities. 12 CHAPTER 1
INTRODUCTION AND LITERAWRE REVIEW
History and Definitions
The use of hydroponics, a term used to describe the cultivation of plants grown in soilless culture, started in Europe in the 17th century. This method of growing plants was first used experimentally to grow herbs and was later expanded to include flowers, foliage, and vegetable crops (Dalrymple, 1973). Near-commercial use of hydroponics was first described by Gericke in 1940 wilo coined the term hydroponics. The use of hydroponics has increased along with the increase in greenhouse plant and vegetable production (Jensen and Collins, 1985).
Hydroponics is a method of growing plants in nutrient solution either with or without the use of an artificial growing medium to provide mechanical support (such as, sand, gravel, rockwool, peat, vermiculite or sawdust). Hydroponic systems which incorporate a growing medium are termed aggregate systems. In liquid hydroponic systems, the roots grow either directly in the nutrient solution or in water-saturated air. An open hydroponic system delivers the nutrient solution to the plants and is not reused. Systems which involve recirculation of the nutrient solution are called closed hydroponics (Jensen and Collins, 1985). More and more, commercial growers are turning to the use of recirculating systems. This is due in large part to environmental concerns regarding ground water contamination and pending legislation in both Europe and the United States which will make it illegal to dispose 13 of greenhouse runoff water. This means that all water, whether used for pot cultures
or hydroponics, will need to be collected and recycled (Blom, 1990).
Growing plants hydroponically in a controlled environment has several
advantages over growing plants in soil, ie - more efficient use of water and fertilizers,
crop production where no suitable soil exists, maximum crop yield in high density
planting, indifference to climatic factors and growing season, minimal use of land, suitability for mechanization, and rapid turnaround of crops. In addition, there is no
need for some labor intensive activities such as soil sterilization. Other presumed
advantages to these systems, which isolate plants away from soil, include less root disease, no salinity problems, and the elimination of poor soil structure and drainage situations (Jensen and Collins, 1985).
Disadvantages of hydroponics over traditional open-field agriculture are largely related to the high costs of capital and energy inputs required for greenhouse cultivation. Thus, the types of crops grown hydroponically are restricted to those of high economic value, to crops which are specific to geographic regions or are seasonal. In the United States cost effective hydroponic production is limited to high-quality vegetables such as tomatoes, cucumbers and specialty lettuce. These vegetables, along with eggplant, melons, peppers, strawberries and herbs are grown in commercial hydroponic systems in Europe and Japan (Jensen and Collins, 1985).
Types of Hydroponic Systems
There are many different types of systems which can be used to grow plants hydroponically. The trend in hydroponic vegetable production is moving toward 14 liquid systems which employ recirculating nutrient solutions. Therefore, this discussion will focus on systems of this nature. The raceway method involves growing plants in a large volume of nutrient solution (Jensen, 1980; Massantini, 1976). The nutrient solution in the raceways does not recirculate, but it is monitored for nutrient levels, replenished and aerated. The flotation boards move through the raceways, and the plants, in the boards, reach maturity as the boards arrive at the end of the raceway. The flotation board is then removed from the system, the plants are harvested and newly planted boards are placed back at the start of the raceway. Another system referred to as the nutrient film technique (NFl') was developed in the late 1960's (Cooper, 1967; Windsor et aI. 1979). In the NFf system, a small volume of nutrient solution flows past plant roots in plastic lined troughs.
The troughs close around the base of the plants to prevent evaporation and to exclude light. The main advantage of the NFl' over other methods is that the volume of the nutrient solution used is greatly reduced. The nutrient solution in hydroponic systems is often treated (i.e. heated or cooled) in order to achieve optimum plant growth and yield. Smaller volumes of nutrient solutions may be more easily treated than the larger volumes of solution used in other systems (Graves,
1983; Hanger, 1982; Jensen and Collins, 1985; Moorby and Graves, 1980; Orchard,
1980).
Several modifications to the traditional NFl' system have been developed in recent years. One example is a system with permanent channels made out of concrete (Jensen, 1983; lauder, 1977). Another modified NFl' system with moveable 15 channels was first developed by Prince, et aL (1981). Moveable channels can be used to give plants more room as they mature allowing for maximum yield potential to be reached and can be more efficient in the utilization of greenhouse space and in mechanization of cultivation (GiacommeIIi et aI., 1982; Schippers, 1982; Varley and Burrage, 1981; Van Os, 1983). Other modifications include the use of stacked channels, moving belts, and vertical systems (Morgan and Tan, 1982; Rogers, 1983;
Schippers, 1978; White, 1980). All modifications to the original NFT system are designed to improve crop production efficiency. The NFl' system along with its modifications is the most common type of system used today (Jensen and Collins,
1985).
Aeroponics is a unique type of hydroponic system in which plant roots are suspended in midair in an enclosed container and are periodically misted with a nutrient solution (Jensen and Collins, 1985; Massantini, 1973). Though not economically feasible for commercial vegetable production, small units are manufactured for home use. Additionally, aeroponics may be worthwhile as a system for rooting cuttings of greenhouse foliage crops (Jensen and Collins, 1985).
Disease Problems in HydTQPonically
Grown Vel:etables
One of the proposed advantages to growing plants hydroponically is the presumed avoidance of soil-borne root-infecting pathogens. However, hydroponic culture is not devoid of disease problems. While growing plants in controlled environments may reduce the diversity of infectious diseases, the incidence and 16 severity of those that do occur can be greater than in open-field agriculture. In
traditional open-field agriculture, rapid development of plant disease is a
characteristic of aboveground disease agents (Agrios, 1988). However, growing plants
in recirculating hydroponic systems now imparts this characteristic to below-ground
root-infecting pathogens. For example, when spinach is grown hydroponically it is
. highly susceptible to Pythium aphanidermatum (Edson) Fitsp. and Pythium dissotocum
Drechs. (Bates and StanghelIini, 1984), yet these pathogens in the field generally
cause little or no disease. Once a pathogen is introduced into a recirculating system,
several factors, such as the abundance of a genetically uniform host, a constantly
favorable environment, and a rapid and effective means of dispersal of the infectious
agent in the recirculating water aid in the uniform distribution of the organism throughout the entire system. Once a pathogen becomes established in a hydroponic
system, control of the disease agent is difficult.
Many pathogens, which infect both foliage and roots have been reported to
occur on hydroponically-grown vegetables. However, foliar diseases caused by
bacteria, viruses and fungi, are not aided in spread or infection by this cultural
technique. A possible exception to this statement has been reported by Paludan
(1985) who experimentally showed that cucumber green mosaic virus and tomato
mosaic virus were spread by the recirculating water. However, virus spread in
recirculating water has not been responsible for serious disease problems in
commercial hydroponic vegetable production. The most serious problems which
occur in hydroponics are caused by root-infecting fungi which are aided by the unique
features of hydroponic culture, i.e. - recirculation of pathogen infested water. 17 Table 1 lists fungal root pathogens which have been isolated from roots of
hydroponically-grown vegetables. Though reported, the pathogens listed in this table
have not been proven to cause disease problems in large scale vegetable production.
Table 2 lists pathogens which are known to cause serious economic disease problems
in commercially-grown hydroponic vegetables. Zoosporic soil-borne organisms, such
as Pythium, Phytophthora, Plasmopara, and Olpidium are the most
common and serious pathogens which occur in hydroponic systems. Zoospores
produced by these pathogens are well suited to operate in the aquatic environment.
Zoospores are motile, asexual, unicellular, propagating or disseminating bodies
produced inside a sporangium (or vesicle) which, upon release, become a free
swimming stage in the life cycle of the fungus. Depending on the species, zoospores
range in size from 3-12 J,lm in diameter. Duration of motility is also dependent upon
the species, but generally ranges from 4-24 hrs (Carlile, 1983; Hendrix and Campbell,
1983; Lange and Olsen, 1983).
Zoospores primarily locate roots through a chemotactic mechanism (Carlile,
1983; Dukes and Apple, 1961; Royal and Hickman, 1964; Lange and Olsen, 1983).
The organism then encysts, penetrates and infects the plant. Under optimum conditions, attraction, penetration and infection can occur within 5 min (Goldberg et aL, 1989).
Furthermore, zoosporic organisms have a tremendous reproduction capacity.
For example, 1 em of a lettuce root infected with Plasmopara lactucae-radicis Stang.
& Gilbn., a newly described root pathogen of lettuce (Stanghellini and Gilbertson,
1988), produces approximately 40 sporangia. Each sporangium produces about 100 18
Table L Root-infecting fungi isolated from roots of vegetables grown in recirculating hydroponics. Pathogen Host References Pythium debaryanum Tomato Jenkins and Averre. 1983; Vanachter, Van Wambeke and Van Assche. 1983. Cucumber Jenkins and Averre. 1983. Lettuce Jenkins and Averre. 1983 Pythium u/timum Tomato Jenkins and Averre. 1983; Vanachter, Van Wambeke and Van Assche. 1983. Cucumber Evans. 1979; Jenkins and Averre. 1983 Lettuce Jenkins and Averee. 1983 Pythium myrioty/um Tomato Jenkins and Averre. 1983 Cucumber Jenkins and Averre. 1983 Lettuce Jenkins and Averre. 1983; Schuerger and Pategas. 1984 Pythium irregu/are Lettuce Schuerger and Pategas. 1984 Pythium sy/vaticum Tomato Vanachter, Van Wambeke and Van Assche. 1983 Phytophthoro cryptogea Cucumber Price and Fox. 1986 PhytophtllOro nicotianae Tomato Evans. 1979; Holderness and Pegg. 1986; Skadow, Schaffrath, Gobler, Drews, and Lanckow. 1984; Staunton and Cormican. 1978; Van Voorst, Van Os and Zadoks. 1987; Vanachter, Van Wambeke and Van Assche. 1983. Cucumber Price and Fox. 1986 Fusarium oxysporum f.sp. Tomato Vanachter, Van Wambeke and Van Assche. 1983. rodicis-/ycopersici Fusarium oxysporum f.sp. Cucumber Jenkins and Averre. 1983. cucumerinum Pyrellochaeta /ycoperisici Tomato Couteaudier and A1abouvette. 1981; Staunton and Cormican. 1978 Collectotrichum coccodes Tomato Couteaudier and A1abouvette. 1981; DaUghtrey and Schippers. 1980; Jenkins and Averre. 1983. Spongospora subterranea Tomato Davies. 1980. Didymedlla Iycopersici Tomato Davies. 1980; Evans. 1979; Staunton and Cormican. 1978; Staunton and Cormican. 1980. Verticillium dahliae Tomato Evans. 1979 Verticillium tricorpus Tomato Ewart and Chrimes. 1980. 19
Table 2. Root-infecting fungi reported as pathogens of vegetables grown in recirculating hydroponics.
Pathogen Host References Pythium apllanidennatum Tomato Jenkins and Averre. 1983; Skadow, Schaffrath, Gobler, Drews and Lanckow. 1984; Stanghellini and Russell. 197L Cucumber Jenkins and Averre. 1983; Nowicki. 1985; Skadow, Scbaffrath, Gobler, Drews and Lanckow. 1984. Spinach Bates and Stangheliini. 1984; Gold and Stanghellini. 1985. Pythium intennedium Cucumber Stanghellini, White and Tomlinson. 1987. Pythium dissotocum Lettuce Stanghellini and Kronland. 1986. Spinach Bates and StangheUini. 1984; Gold and Stanghellini. 1985. Phytophthora cryptogea Tomato Holderness and Pegg. 1986 Lettuce Linde, Stanghellini and Matheron. 1990. Phytophthora nicotianae Tomato Skadow, Schaffrath, Gobler, Drews and Lanckow. 1984; Van Voorst, Van Os and Zadoka. 1987. Plasmopara lactucae-radicis Lettuce Stanghellini, Adaskaveg and Rasmussen. 1990; Stanghellini and Gilbertson. 1988 Fusarium oxysporum f.sp. Tomato Couteaudier and Alabouvette. 1981; Evans. 1979; Iycopersici Staunton and Cormican. 1978. Oplidium radicale Cucumber Tomlinson and Thomas. 1986. Olipidium brassicae Lettuce Tomlinson and Faithfull. 1980. 20 zoospores. A uniformly infected lettuce plant, with approximately 2,000 cm of roots,
will produce about 8 million zoospores (Stanghellini, unpublished).
In liquid hydroponics, there are no barriers (such as soil particles) to impede
the movement of zoospores. Therefore, zoospore release results in rapid infection of all plants within the system. The greater number of zoospores in a hydroponic
system, the greater the extent of the damage. Accordingly, zoospores play an
important role in the spread of these fungi.
In addition to spread of the fungus, some zoosporic organisms have the ability
to transmit other pathogens. For example, Olpidium brassicae (Wor.) Dang. and
Olpidium radicale Schwartz & Cook are the vectors of lettuce big-vein virus
(Tomlinson and Faithfull, 1979) and tobacco necrosis virus (Paludan, 1985); and melon necrotic spot virus (Tomlinson and Thomas, 1986), respectively. Zoospores
of these fungi carry the virus (or virus-like agent) and upon infection of the roots
transmit the virus to the plant (Campbell, 1962; Campbell and Grogan, 1963;
Komuro, et. al., 1970; Tomlinson and Garrett, 1962, 1964; Vetten, et. al. 1987).
Although these viruses are typically problems in soil-grown vegetables, facilitation of zoospore spread in hydroponics has enabled these viruses to become serious disease problems in Europe in greenhouses employing NFl' systems (Cooper, 1975;
Tomlinson and Faithfull, 1980; Tomlinson and Thomas, 1986).
Disease Symptoms
Critical in the control of plant disease is realization that disease problems exist. Some of the pathogens listed in Table 2 cause distinct symptoms such as root 21 rot, wilt, reduced plant growth and/or fruit yield, and, in some cases, plant death.
For example, hydroponically-grown spinach is highly susceptible to two species of
Pythium, P. aphanidermatum and P. dissotocum (Bates and Stanghellini, 1984).
Infected plants express symptoms of severe root rot, wilt and stunting, and the plants
die within 1 wk after transplanting. As a result of the devastating nature of these pathogens, commercial hydroponic spinach production in Arizona was abandoned.
Additionally, some of the organisms listed in Table 2 cause diseases in which
there are no obvious symptoms. The roots and shoots are normal in appearance, yet the growth rate and yield of the plants are significantly reduced. These subclinical diseases are more difficult to detect as the nature of hydroponic culture leads to uniform infection of all plants in the system. Thus, all plants appear healthy, yet they are actually diseased. An example of the occurrence of a subclinical disease in hydroponics was discovered by Stanghellini and Kronland (1986) who attributed significant reduction in yield of lettuce to infection by P. dissotocum. Pythium dissotocum asymptomatically infects the feeder rootlets and occupies about 75% of the total root mass. At optimum temperatures for the fungus, yield losses of up to
54% were recorded.
Obviously, regardless of whether an organism produces clinical or subclinical disease symptoms, control is essential for optimum crop yield. One of the first steps towards achieving effective control is identification of the pathogen (to species if possible) because the control method of choice depends on the specific pathogen involved (StangheIlini, 1988). 22 Introduction and Spread of Pathogens
Identification and elimination of the source(s) of pathogen introduction and spread within greenhouses and hydroponic systems is of utmost importance in
control. Potential sources of pathogen introduction and spread in recirculating
hydroponic systems, include seed or plant material, water, soil, sand, peat, and insects. Although none of the root-infecting pathogens listed in Table 2 are known to be carried in or on seed, other organisms (e.g. bacteria and viruses) which cause foliar diseases have the potential to be introduced to greenhouses in this manner.
Corynebacterium midziganense (E.F. Sm.) H. L. Jens, a pathogen reported to occur in soilless culture (Davies, 1980; Couteaudier and Alabouvette, 1981), is seed-borne.
However, this pathogen has not caused large scale disease problems in hydroponics and is not spread in NFT systems (Davies, 1980). Currently, only lettuce mosaic virus is known to have been introduced into greenhouses by seed (Brown and
Stanghellini, unpublished). Control for this disease consists of using virus-free seed.
In addition to seed, infected plant material is another potential source of pathogen introduction. Thus, maintenance of disease free stock is essential in the establishment and protection of pathogen free systems.
Irrigation water is a likely source for the introduction of pathogens into hydroponic systems. Reservoirs, rivers and streams are known to be infested with many plant pathogens, including several of the organisms listed in Table 2.
Development of effective water treatment for the elimination of plant pathogens would be beneficial in controling diseases in recirculating hydroponics. 23 Even though the definition of hydroponics is essentially "soilless culture", soil
particles are still a source of pathogen introduction into hydroponic systems. Soil is
continually brought into facilities on the shoes of greenhouse personnel. In addition,
sumps or reservoirs used to hold nutrient solutions are often located at ground level
where accidental introduction of soil into the solution can easily occur. While
elimination of soil from shoes is probably not possible, construction of physical
barriers around sumps or reservoirs can help to reduce accidental introduction of soil
into the system.
Pathogenic organisms, such as P. aphanidennatum and P. dissotocum, have
been isolated in Arizona from river sand. Washed river sand has been implicated
as the source of introduction of these two species into a commercial hydroponic
facility in Arizona. The sand was used throughout the facility in walkways between
hydroponic raceways (Bates and Stanghellini, 1984).
Peat is often used in small amounts to help anchor young seedlings in hydroponic systems (Jensen and Collins, 1985). Yet, 52 commercial peat products surveyed by Kim, et. aL, (1975) contained Fusarium spp. and 15 of the products contained various species of Pythium. Likewise, 10 of 12 peat products tested in
Canada were infested with Pythium (Favrin, et aL, 1988). Furthermore, peat is suspected as the source of introduction of Olpidium spp. (Tomlinson and Faithfull,
1980; Tomlinson and Thomas, 1986), and Fusarium oxysporum Schlecht f.sp. lycopersici Sacco (Couteaudier and Alabouvette, 1981) in hydroponic facilities in
Europe. 24 Insects have long been suspected as carriers of disease agents, but proof of introduction of root-infecting pathogens into greenhouses or hydroponic systems by this means is lacking. Insects have, however, been implicated in the spread of root pathogens within greenhouses. Recently, Kalb and Millar (1986) reported that externally infested adult fungus gnats (Bmdysia impatiens Johannsen) experimentally disseminated Verticillium albo-atrum Reinke & Berthold to soil-grown alfalfa plants in growth chambers and greenhouses. This insect is also suspected as a potential vector of Pythium spp. in greenhouses (Favrin, et. aL 1988). Their potential to function as vectors for Pythium was strengthened by the recent discovery that laboratory-reared larvae of fungus gnats, fed on pure culture of Pythium spp., ingest and excrete germinable zoospores (Gardiner, et. aL, 1990). Although introduction of root-infecting fungi into greenhouses and hydroponic systems by insects has not been documented, lettuce infectious yellows virus was introduced into a commercial hydroponic facility in Pennsylvania by white flies (Bemesia tabaci Genn.) (Brown and
StangheIIini, 1988).
In addition to spread by insects, pathogens can move from plant to plant within a hydroponic system by a number of methods. Unique features of hydroponic systems facilitate dispersal of microorganisms. Fungal spores are aided in movement throughout the system by the recirculating nutrient solution. For example, deciduous sporangia produced by Pythium intermedium de Bary are rapidly and uniformly spread throughout the system (Stanghellini, et. aL, 1988). When these sporangia come in contact with roots they are capable of direct penetration and infection or they may produce zoospores. Recycling solutions may assist fungal zoospores and 25 bacteria in locating receptive infection sites by providing quick and unobstructed access to all roots in the system. Furthermore, root-to-root spread of fungi is facilitated in liquid hydroponic systems, especially in NFl' systems, where plants are grown in close proximity to one another. These methods of spread probably occur simultaneously.
Methods of Disease Control
There are three general methods for controlling plant diseases. The use of
(1) chemicals, (2) cultural and physical practices, and (3) biological methods.
Control of diseases in hydroponics is difficult as some control methods available for cultivation in soil are unavailable or ineffective for use in hydroponics. For example, there are no fungicides currently registered in the United States for use in hydroponically-grown vegetables. In addition, sanitation through the removal of diseased plant material is impractical because once the pathogen establishes itself in the hydroponic system, all of the plants are infected.
Although hydroponics lends itself to easy application of chemicals, other features of hydroponic culture prohibit the use of pesticides in the system. The use of many chemicals in food crops requires 14 days between application and harvest.
Yet most commercial hydroponic systems are designed to produce harvestable plants everyday (Jensen and Collins. 1985). In addition, pesticide build up on fruit and leaves can reach intolerable levels in hydroponically-grown plants. And, lastly, development of resistant strains of the pathogen may be facilitated by constant exposure to the chemical. 26 The use of surfactants is an attractive alternative to the use of microbial pesticides. Zoospores of Pythium spp., Phytophthora nicotianae Van Breda de Haan
(Stanghellini and Tomlinson, 1987), Olpidium brassicae (Tomlinson and Faithfull,
1980) and Olpidium radicale (Tomlinson and Thomas, 1986) are highly sensitive to non-ionic surfactants. Zoospores exposed to the substance cease motility and lyse within a few minutes. Although not registered for use in the United States, Agral (leI Plant Protection Division, Fernhurst, Haslemer, Surrey, United Kingdom) has been used successfully in Europe to control viruses vectored by zoospores of
Olpidium (Tomlinson and Faithfull, 1980 and Tomlinson and Thomas, 1986). One limitation to the use of surfact ants is that fungal propagules which have a cell wall
(e.g. hyphae, encysted zoospores, and oospores) are immune to the lytic action of the surfactant and as such are unaffected by the material (Stanghellini, unpublished). Cultural or physical methods of control, such as sanitation and pest management, treatment of infested water, and manipulating the environment, have potential for control of some infectious agents in hydroponics. However, as with chemical control there are limitations.
Sanitation and effective pest control are essential in the maintenance of a pathogen free system, yet, as mentioned, removal of infected plants is difficult when all plants are infected. Additionally, disinfestants, such as chlorine, though commonly used to disinfest equipment, are not registered for use in greenhouses. Likewise, adequate control of insects can be difficult to achieve.
Elimination of pathogens by treatment of the recirculating nutrient solution has been proposed as a strategy for control of diseases in hydroponics (Stanghellini, 27 1988). Methods of water treatment include filtration, ultraviolet irradiation,
sonication, thermal inactivation, and ozonation. Though proposed as potential
control methods, there is currently little scientific evidence that treatment of water
to eliminate microorganisms will be effective at the commercial level. Ultimately,
effective control by these means will be dependent on two factors. First, it will be
dependent on the specific pathogen involved. For example, zoospores of Pythium
and Phytophthora, which are 8-12 JDD, may be removed by filtration through a 5-7 JIm
filter. However, bacteria, viruses and zoospores of Olpidium spp. (which are
approximately 3 J.lm) would readily pass through the same filters. Secondly, efficacy
will be dependent on dwell time and flow rate of the nutrient solution in the facility.
For example, StanghcIIini, et al. (1984) demonstrated that ultraviolet light could be
used to eliminate zoospores of P. apizanidermatum from infested nutrient solution at
a flow rate of 124 L/min, yet control was not achieved at a flow rate of 300 L/min.
A similar situation would probably occur with the use of sonication, thermal inactivation and ozonation. However, these control methods may still be of use in the nursery area of a commercial operation where the volume of nutrient solution used is greatly reduced.
The manipulation of the environment is another possibility for the control of root disease in hydroponics. As mentioned previously, the relative ease of manipulation of water and/or air temperatures in hydroponics is an advantage of this type of culture over field agriculture. The pathogens listed in Table 2 have optimum temperatures at which they cause disease. For example, P. apizanidermatum is a high temperature pathogen which is most damaging in water temperatures over 24 C 28 (Bates and Stanghellini, 1985). In contrast, P. dissotocum (Bates and Stanghellini,
1984; Gold and Stanghellini, 1985) and Olpidium spp. are favored by water
temperatures below 24 C. Additionally, the optimum water temperature for
Plasmopara lactucae-radicis ranges from 23-26 C (StangheIJini, et ai, 1990). Thus,
raising and lowering the temperature of the nutrient solution can be effective in
decreasing the activity of certain fungi.
One of the more exciting strategies for control is the use of biological methods such as resistant varieties and beneficial microorganisms. Again, identification of the pathogen to species will be essential for effective control. Some cultivars may be
resistant to a particular strain of the pathogen. Resistant cuItivars have been used
to effectively control two of the pathogens listed in Table 2. Tomato cultivars
resistant to Fusarium oxysporum f. sp. lycopersici are commercially available.
Additionally, 'Sitonia', a cultivar of leaf lettuce, is resistant to Plasmopara lactucae radicis, and can be grown during the summer production months when temperature
of the nutrient solution favors the fungus. In the winter, when the fungus is inactive,
commercially preferable yet susceptible cuitivars such as 'Ostinata' and 'Salina' can
be cultivated (StanghelIini, et al., 1990).
For many years, the use of beneficial microorganisms has been touted as the future in plant disease control. However, there has been little progress in the development of commercially available biological control agents. One problem in achieving effective control relates to specificity of the control agent. Microorganisms used as antagonists may inhibit growth or activity of one species, but not of another, even closely related, organism. Another problem in effective control lies in 29 competition between the beneficial agent and resident microorganisms in the
rhizosphere. Cultivation in water may ultimately be the best cultural system for the
effective use of biological control agents. In hydroponics the control agent could be
spiked into the system at the time of planting allowing for colonization of the
rhizoplane by the beneficial organism prior to introduction of the pathogen. There
are currently only two studies in which microbial factors have been examined as a
control method for hydroponically-grown vegetables. Couteaudier and Alabouvette
(1981) added a small amount of soil, naturally supressive to Fusarium wilt pathogens, to peat used to rear tomato seedlings for cultivation in a Nfl system. In treatments where 5% of the supressive soil was added to the peat, only 8% of the plants became diseased. In contrast, 100% of the plants were diseased in unamended controls.
Anderson and Guerra (1985) demonstrated that beans germinated in vermiculite in the presence of Pseudomonas putida (Trevisan) Migula and subsequently transferred to a hydroponic growing system were colonized on all root surfaces by the bacterium.
Bacterial colonization of the roots was maintained at constant level throughout an
18 day growth period. Colonization by this bacterium provided delayed protection from Fusarium solani (Mort.) Sacco f.sp. phaseoli (Burk.) Snyd. and Hans., however, it should be noted that beans are not commonly cultivated in hydroponics and
Fusarium solani f.sp. phaseoli has not been reported to occur in hydroponic systems.
To date there has been no report where beneficial microorganisms have been added to the nutrient solution.
Although control of diseases in recirculating hydroponics is difficult, it is not impossible. The more that is known regarding the epidemiology and biology of the 30 pathogens involved, the easier it will be to develop adequate control methods. Ultimate control of diseases in hydroponics will most likely come from the integrated use of a number of the above control strategies.
Objectives
The objectives of the studies reported herein were to investigate the potential use of filtration as a control strategy for P. aphanidermatum in recirculating hydroponics and to investigate shore flies (Seatella stagnalis Fallen) as a potential vector of the pathogen into hydroponic systems. 31 CHAPTER 2
FILTRATION AS A STRATEGY FOR CONTROL OF PITH/UM
APHAN/DERMATUM IN RECIRCULATING HYDROPONICS
One of the most common pathogens causing root disease in hydroponically-grown vegetables is Pythium aphanidennatum Edson (Fitzp.). Once introduced into a recirculating hydroponic system, rapid and uniform dispersal of the pathogen occurs results in infection of all plants within the system. Devastating crop losses have been reported to occur on tomatoes (Jenkins and Averre, 1983; Skadow et at., 1984;
StangheIIini and Russell, 1971), spinach (Bates and StanghelIini, 1984; Gold and
Stanghellini, 1985), and cucumbers (Jenkins and Averre, 1983; Skadowet aL, 1984).
Experimentally, metalaxyl is effective in controlling Pythium spp. in hydroponics
(Bates and StangheIIini, 1984; Gold and StangheIIini, 1985), however, this material is not registered for use in this type of cultural system. Ultraviolet irradiation can be used to eliminate zoospores from recirculating nutrient solutions, although effective control of the pathogen is dependent on the volume of water treated and the flow rate of the solution through the UV unit (StangheIIini, et at., 1984). This method of control was not found to be effective when a flow rate similar to that used in commercial facilities was examined. Filtration of the nutrient solution to physically remove zoospores from the system has been suggested to have potential as a method of control (StangheIIini, 1988). However, experimental evidence of the effectiveness of this method is lacking. 32 The objective of this research is to investigate the potential use of filtration as a strategy for control of P. aphanidennatum in recirculating hydroponics.
Materials and methods
Three separate above ground hydroponic cultural tanks each measuring 2.4
X 1.2 X 0.5 m were used (Fig 1). Each tank contained 850 L of a complete, aerated, nutrient solution. The nutrient solution consisted of Hydrosol (a Peter's fertilizer product, W. R. Grace and Co., FoelsvilIe, Pennsylvania), magnesium sulfate, potassium nitrate, and calcium nitrate. The fertilizer was added to each hydroponic tank at the rate of 972 g Hydrosol, 200 g magnesium sulfate, 544 g potassium nitrate, and 875 g calcium nitrate. The temperature of the nutrient solution was maintained at 30 C.
Cucumber (Cucumis sativus L. 'Toska 70') seeds were germinated aseptically on 2% water agar in petri dishes and transplanted in Oasis horticubes (Smithers
Oasis, Kent, Ohio). The seeded cubes were placed in holes cut in styrofoam flotation boards (2.1 X.9 X 0.03 m). Each board held 21 seedlings spaced 30 cm apart (Bates and StangheIIini, 1984). The boards were floated on top of the nutrient solution in the hydroponic tanks. Pythium aphanidennatum inoculum was introduced into tank
1 on two infected plants, as follows: 2 plants were removed from the tank and the roots were placed in a suspension of P. aphanidennatum zoospores. After 1 hr, the plants were returned to tank 1. These plants served as a source of inoculum for the 33
Figure 1. Recirculating hydroponic system. A, diagrammatic representation and B, photograph of cultural system used in the study of the efficacy. of filtration on the control of root rot of cucumber caused by Pythium aphanidennatum. 34 remaining plants in the tank. The plants were 3-wk-old at the time of inoculation.
In a repeat experiment, the plants were 1-wk-old at the time of inoculation.
Six days after inoculation, the infested nutrient solution was recirculated at a
flow rate of 114 L/min for 30 min (5X turnover) as follows: infested nutrient solution was first passed through a 20-J.lll1-cartridge filter (Model CFf25 with C5625 filter,
Jacuzzi, Little Rock, Arkansas); one portion of the latter volume (about 57 L/min) was diverted into tank 2 and returned to tank 1, and a second portion (about 57
L/min) was diverted through a 7-Jlm-cartridge filter (Model PCY1RFG 16 with
R1F070 filter, Pall Process Filtration Corporation, East Hills, New York) into tank
3 and also returned to tank 1 by a submersible pump. Recirculation was conducted every other day for 5 days.
Root samples were collected from plants in all tanks 24 hr after each recirculation of the nutrient solution. Ten 2-cm-Iong root segments per plant were blotted dry on paper towels and plated on 2% water agar amended with 200 JJg/ml streptomycin sulfate. The plates were incubated at 37 C for 24-48 hr. Mter the 3rd and final run of the filtration system, the surface (0 nun), middle (8 nun depth) and inner core (16 mm depth) of the 7-J.lll1 filter was sampled for the presence of the
2 pathogen (Fig 2). Twelve pieces, each 15 nun , were plated on 2% water agar amended with 200 JJg/ml streptomycin sulfate and selective media (Burr and
Stanghellini, 1973) and incubated at 37 C for 24-48 hr. Fungi isolated from either the roots or the filter were subcultured on V-8 juice media and/or selective media and identified to species. The experiment was repeated once. 35
Figure 2. Photograph of the 7-J.l.m filter cartridge used to study the efficacy of filtration of zoospore infested water in the control of root rot of cucumber caused by Pythium aphanidennatum. a-c. various sampling depths for the attempted isolation of the pathogen. a = surface (0 mm), b = middle (8 mm depth), c = inner core (16 mm depth). 36 Results and Discussion
Six days after infestation of tank 1 (the start of the recirculatory study), all cucumber plants in tank 1 were dead (Fig. 3A) and P. aphanidermatum was isolated from all roots sampled from tank 1 (Fig 4A). One day after initiation of recirculation, the fungus was isolated from 67% of the plants in tank 2 which received infested nutrient solution from tank 1 that had passed through the 20-J.lm filter. All of the plants in tank 2 were infected 3 days after initiation of recirculation, the plants were beginning to wiIt (Fig. 3B) and all plants died within 6 days. After initial recirculation, the fungus was not recovered from any plants in tank 3 which received 7-J.lm filtered water and the plants appeared healthy (Fig. 3C). However, the fungus was isolated from 1 plant (2% of the plants) in tank 3 after 2 consecutive recirculation cycles and all plants were infected within 7 days. A repeat experiment gave similar results. However, the onset of infection was delayed (Fig 4B). In this case, 60% of the plants in tank 2 were infected 7 days post-recirculation. In tank 3,
1 plant (10%) was infected 10 days post-recirculation. All plants in tanks 2 and 3 became infected within 14 days. The delay in the onset of disease (between the two experiments) is presumed to be due to the age difference between the plants at the time of inoculation. The younger seedlings in experiment 2 had a smaller root system and consequently produced less inoculum for infection. The speed in which plants in the treated tank (tank 3) became infected after introduction of P. aphanidermatum illustrates one of the key problems in control of pathogens in 37
, ,(
Figure 3. Disease incidence in cucumbers resulting from the recirculation of water from (A) tank 1, which was artificially infested with Pythium aphanidennatum, to (B) tank 2 and (C) tank 3 five days after receiving infested water passed through a 20-J.lm and 7-tJ.m filter, respectively. Figure 4. Efficacy of filtration of infested water to control root rot of cucumber caused by Pythium aphanidermatum in a recirculating hydroponic system. Plants in tank 1 were inoculated on day O. Arrows indicate days of recirculation. A Experiment 1 (plants were 3 weeks old at time of inoculation). B. Experiment 2 (plants were 1 week old at time of inoculation). 39 recirculating hydroponics: rapid and uniform spread of inoculum and infection by the
pathogen.
Pythium aphanidermatum was recovered from the surface and the middle, but
not from the inner core, of the 7-J..lITl filter (Table 3). From these results it appears
that the filter was effective in removing the zoospores from the nutrient solution.
Since the plants in the 7-J.lm treated tank eventually became infected, other sources of pathogen introduction and spread into the system were investigated (see
Chapter 3). It was discovered that shore flies (Seatella stagnalis Fallen) are an effective aerial vector of P. aphanidermatum. This insect was determined to be the probable cause of contamination by the fungus in the treated tank. Attempts to eliminate the flies in the experiment were undertaken. A clear plastic tarp was draped over the entire tank, holes in the tarp allowed for the seedlings to emerge and the base of the seedlings were painted with grease. Despite these attempts, the fungus entered the system. Although the flies were not documented as the source of contamination in the 2nd experiment, they are presumed to be the source of infestation because total exclusion of insects was not possible and P. aphanidermatum was not recovered from the inside of the filter.
Greenhouse hydroponic systems which utilize a reservoir for recirculating nutrient solution to and from growing chambers are common in commercial vegetable production. Upon entering the system at any location, pathogens are rapidly disseminated to all plants. Fortunately, reports of serious disease problems 40
Table 3. Recovery of Pythium aphanidermatum from the 7-f-Im filter cartridge8
------.---_.---_.------
Filter Depth Experiment 1 Experiment 2
Surface (0 mm) + + Middle (8 mm) + +
Inner Core (16 mm)
2 8 Recovery of P. aphanidennatum from twelve-15-mm pieces of filter material plated on 2% water agar or Pythium selective medium. + 100% recovery 0% recovery. 41 in the industry are rare. However, devastating crop losses caused by P.
aphanidermatum have occasionally been reported (Bates and Stanghellini, 1984; Gold
and Stanghellini, 1985; Jenkins and Averre, 1983; Skadow, et al., 1984; Stanghellini
and Russell, 1971) and, due to lack of effective control measures, cultivation of the
susceptible crop has been abandoned (Bates and Stanghellini, 1984; Gold and
Stanghellini, 1985). Results of this study indicate that filtration through a 7-J-lITl filter
is effective in removing zoospores of P. aphanidennatum from nutrient solution.
However, the use of this strategy as a control method in commercial hydroponic
facilities is impractical. Several factors prohibit the feasibility of using this system.
First, zoospores of other Pythium spp. and Phytophthora spp., which are of similar size, would probably be removed by this system. However, zoospores of Olpidium spp., which are 3-5 J,.tm in diameter, and viruses would easily pass through a 7-J,.tm filter. Thus, filtration would be ineffective in the control of diseases caused by these pathogens. Second, the maximum flow rate through the 7-J-lITl filter is 56 L/min.
However, commercial facilities generally pump large volumes of nutrient solution
(378.5 L/min). Third, the pore size of the filter leads to rapid clogging by debris, such as algae, root and plant material, soil, and sand. As the filter clogs up with debris, the flow rate is reduced. Eventually, the flow of nutrient solution through the filter is stopped completely. 42 CHAPTER 3
INGESTION-EGESTION AND AERIAL TRANSMISSION OF
PYTHIUM APHANIDERMATUM BY SHORE FUES
(EPHYDRINAE: SCATELLA STAGNALIS)
One advantage to growing plants hydroponically is the presumed avoidance of root diseases; however, hydroponic cultural systems are not devoid of disease problems. The most common root diseases that occur in hydroponic culture are caused by various species of Pythium (Bates and Stanghellini, 1984; Jenkins and
Averre, 1983; Stanghellini and Kronland, 1986; Stanghellini and Russell, 1971).
Determination and elimination of the source(s) responsible for the introduction of these root pathogens into commercial greenhouses are requisite for the development of effective disease control.
During the course of my investigations on nonchemical methods for the control of Pythium root rot of cucumbers (Cueumis sativus L. 'Toska 70') cultivated in a recirculating hydroponic system, infection of plants by Pythium aphanidennatum
(Edson) Fitzp. in presumed pathogen-free areas of the system was occasionally encountered. It also was observed that the facility was infested with shore flies
(Ephydrinae: Seatella stagna/is Fallen). Eggs, larvae, pupae, and adult flies (Fig. 5) of this common greenhouse pest, which reportedly feeds primarily on algae (Foote,
1977), were noted in great abundance around the base of cucumber plants. In preliminary studies, P. aphanidennatum was isolated from one out of 13 adult flies 43
Figure S. Various life stages of a shore fly (Seatella stagna/is). A, egg. B, larvae. C, pupae. D, adult. 44 collected in the greenhouse facility. Documentation of the ingestion-egestion and transmission of P. aphanidennatum by larvae and adult shore flies is presented herein.
Materials and Methods
Collection of insects and transmission studies, unless otherwise specified, were conducted in a greenhouse containing an aboveground, recirculating, hydroponic system in which cucumber plants, approximately 3-wk-old, were being grown. Many plants were infected with P. aphanidennatum, and roots of infected plants contained numerous oospores of the fungus (Fig. 6). The recirculating hydroponic system consisted of three interconnected cultural tanks, each containing 850 L of a complete, aerated, nutrient solution. The nutrient solution consisted of Hydrosol (a Peter's fertilizer product, W. R. Grace and Co., Foelsville, Pennsylvania), magnesium sulfate, potassium nitrate, and calcium nitrate. The fertilizer was added to each hydroponic tank at the rate of 972 g Hydrosol, 200 g magnesium sulfate, 544 g potassium nitrate, and 875 g calci!lm nitrate. Plants, 21 per tank and spaced 30 cm apart, were anchored in a styrofoam flotation board, measuring 2.4 X 1.2 X 0.03 m (Bates and
Stanghellini, 1984).
Pathogen infestation of adult flies, pupae, and larvae
To determine the extent of insect infestation by P. aphanidennatum, adult flies were collected daily by aspiration from the greenhouse over a 6-day period. 45
Figure 6. Photomicrograph of oospores of Pythium aphanidermatum in the cortex of a naturally infested cucumber root. Bar = 20 J,Lm. 46 Additionally, larvae and pupae, which were located around the base of infected
plants, were collected daily over a 14-day period. Adult flies, larvae, and pupae were
transported to the laboratory, aseptically squashed, and plated on water agar
containing streptomycin sulfate at 200 J,Lg/ml (Sigma, St. Louis, Missouri). After 48
hr of incubation at 37 C, isolated fungi were identified. The frequency of isolation of P. aphanidennatum from each life stage of the insect was recorded. Additionally, all squashed insect specimens were examined microscopically for direct evidence of the location and nature of fungal structures on or in the various life stages of the insect.
Germinability of oospores recovered from larvae, pupae, and adult flies
To test their germinability, oospores released from squashed larvae, pupae, and adult flies were washed onto a selective agar medium (Burr and Stanghellini, 1973).
After 48 hr incubation at 37 C, the number of colonies of P. aphanidennatum were recorded, and the origin (nature of the propagule) of each colony was microscopically determined.
Germinability of oospores in insect frass
Live larvae and adult flies were collected from around the base of plants in the greenhouse and placed in petri dishes containing selective medium. Within 1 hr, numerous, discrete, frass deposits from larvae and adults were observed in all dishes.
The plates then were placed in an incubator at 37 C. After 48 hr incubation, the 47 origin of colonies of P. aphanidennatum developing on the medium were determined
microscopically.
Larval transmission of P. aphanidennatum
Ten larvae, collected from around the base of cucumber plants in the infested
greenhouse, were placed around the base of a healthy 2-wk-old cucumber plant
housed in a small, hydroponic chamber. The chamber then was sealed with parafilm
having air holes. Control plants were placed in identical chambers with no larvae
added. Mter 4 days at room temperature in the laboratory, the chambers were
transferred to an incubator at 27 C. Mter an additional 3 days, the entire root
system was divided into 4-cm segments. Segments were blotted dry on sterile paper
towels and plated on water agar. Mter 48 hr at 37 C, isolated fungi were identified.
Isolation of P. aphanidennatum from roots was considered as proof of insect
transmission. The experiment was repeated twice with four replicates.
Adult fly transmission of P. aphanidennatum
Transmission studies with adult flies were conducted in the laboratory and in the fly-infested greenhouse. First, 15 adult flies, collected in the greenhouse by aspiration, were immediately placed in each of five separate hydroponic chambers.
Each chamber contained a healthy 2-wk-old cucumber plant. Two additional chambers containing plants without flies served as controls. The chambers were sealed with parafilm having air holes and assessed for transmission as outlined above. 48 Second, seven, 2-wk-old healthy cucumber seedlings, reared individually in portable
hydroponic chambers, were transported to the fly-infested greenhouse. These
chambers were placed randomly throughout the facility. Two of the chambers,
however, were covered with insect-proof cages. Mer 6 days, 10 root segments (each
4 cm in length) from each plant were individually assessed for infection by P. aphanidermatum as described above.
Results
Pathogen infestation of adult flies, pupae, and larvae.
P. aplzanidermatum was isolated from larvae, pupae, and adult flies. However, the level of infestation varied depending on the specific life stage of the insect (Table
4). Oospores, but no hyphae, of P. aphanidermatum were observed microscopically in the gut of squashed specimens of larvae (Fig. 7A-B), pupae, and adult flies.
Additionally, movement of ingested oospores, as well as algal cells, within the gut of live larvae was observed microscopically during peristaltic contractions of the alimentary canal. The number of oospores varied from 25 to over 680 per larva.
Oospores in the gut of infested adult flies ranged in number from 1 to 25 per individual.
Genninability of oospores recovered from larvae, pupae, and adult flies
Oospores in the gut of larvae, pupae, and adult flies appeared to be normal, 49
TABLE 4. Percent of adult flies, pupae and larvae infested with Pythium aphanidermatum8
------.------.------
No. No. Percent Sampled Infested Infested
Adult flies 263 27 10%
Pupae/3rd instar 315 65 20%
Larvae (1st and 2nd instar) 60 58 97%
8 Infestation was documented by isolation on a selective medium and by microscopic observation. 50
Figure 7. Photomicrograph of oospores of Pythium aphanidennatum in the gut of a shore fly larva. A, 4 X and B, 10 X. Oospores are approximately 20J,lm. 51 mature oospores. Oospores that were washed from squashed adult flies, larvae, or pupae germinated readily on selective medium, and developing colonies were traced to germinating oospores. In addition, oospores in squashed larvae were counted and then washed onto selective medium. In three separate washings, 38 of 115, 9 of 36, and 10 of 30 oospores from infested larvae germinated and developed into colonies.
Germinability of oospores in frass
Excreted oospores were observed microscopically in frass deposited by larvae and adult flies (Fig. 8A). Colonies of P. aphanidennatum were obtained on selective medium from both the larval and the adult frass (Fig. 8B), and the origins of the colonies were traced to germinated oospores in frass droplets.
Larval and adult fly transmission of P. aphanidennatum
Pytlzium aplzanidennatum was isolated from the roots of one of five plants in the first adult fly experiment, but not from roots of the control plant. Transmission of the fungus also occurred when larvae were placed around the base of all healthy seedlings.
In the second experiment, adult flies were observed around the base of the uncaged plants within 5 min after placing the individual hydroponic chambers in the fly-infested greenhouse. After 1 day, frass clearly was visible around the base of the plants and on the stem of the cucumber seedlings, and, within 6 days, the fungus was 52
Figure 8. Photomicrograph of oospores of Pythium aphanidennatum from adult fly frass. A, ungerminated and B, germinated oospore from the frass of a naturally infested adult shore fly. Oospores are approximately 20J.lm. al = algal cell. 0 = oospore. og = oogonium. 53 isolated from roots (some of which were necrotic) of all uncaged plants in the fly- infested greenhouse. Pythium aphanidennatum was not isolated from the roots of plants that had been enclosed in insect proof cages. Within 6 days after the cages were removed from control plants, they also were colonized by P. aphanidennatum.
Discussion
Commercial peat-based propagation mixes (Favrin, et al. 1988; Kim, et al. 1975) and naturally infested sand (Bates and Stanghellini, 1984) are primary inoculum sources of various species of Pythium, and other root-infecting fungi in greenhouses.
This study documents, for the first time, acquisition and aerial transmission of P. aphanidermatum by shore flies from infected to healthy plants in a greenhouse.
Oospores of P. aphanidermatum visually were observed in the gut of larvae, pupae, and adult shore flies, and viable oospores were excreted in frass produced by larvae as well as adult flies. Larvae, which fed on infected roots laden with oospores (the only source of oospores in our investigation), acquired the fungus. Because adult flies did not have access to infected roots, the oospores observed in the gut of adult flies likely reflect residual oospore populations retained after pupation. These residual oospore populations, however, were sufficient to infect all plants frequented by naturally infested adult flies.
The presence of fungal propagules in the gut of insects has been reported (Leach,
1940). Thus, any insect stage that feeds upon infested plant material could ingest 54 propagules of any fungus present and could be potential vectors of that organism.
For example, we observed sporangia of Plasmopara lactucae-radicis Stang. & Gilbn., a newly described root pathogen (StangheIIini and Gilbertson, 1988), in the gut of shore fly larvae recovered from roots of infected lettuce plants grown under hydroponic conditions. Additionally, externally-infested, adult fungus gnats (Bradysia impatiens Johansson) experimentally disseminated Verticillium albo-atTUm Reinke &
Berth. to soil-grown plants in a greenhouse (Kalb and Millar, 1986), and this insect also is suspected to be a potential vector of Pythium in greenhouses (Favrin, et al.,
1988). Their potential to function as vectors for Pythium is strengthened by the recent discovery that laboratory-reared larvae of fungus gnats, fed in the laboratory on pure cultures of P. aphanidennatum and other species of Pytlzium, ingest and excrete germinable oospores (Gardiner, et. aI., 1990).
Increased germinability of oospores of P. aphanidennatum after passage through the alimentary canal of snails has been reported (Stanghellini and Russell, 1971).
Whether oospore germinability was altered by passage through the gut of shore flies is not known; however approximately 30% of the oospores recovered from the gut of shore fly larvae were capable of germination.
Shore flies are common pests in many commercial greenhouses and, in light of their newly documented role as a vector of Pythium, strategies for control of infestations by this insect should be implemented. Infested adult flies may account for introduction and spread of Pythium within commercial greenhouse facilities. 55 REFERENCES CITED
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