Bhopal 1986

Madhya Pradesh Vigyan Academy, Fifth Session,

Bhopal 1986 SECTION OF BOTANY

Sectiollal President

PRAKASH SINGH BISEN

Presidential Address

CYANOPHAGE HISTORY AND CONTROL

LIKELIHOOD AS A

Mr. President, distinguished delegates, ladies and gentlemen, First of all, let me ex press my deep sense of gratit ud e to the senior scientists of this state for un anim ously nominat ing me as the Presiden t of Botany section for the fifth annual session of M. P. Vigyan Academy at Bho pal. Viruses have gained increasing acceptance as a promising approach to pest control. Under favourable conditions these viruses provide a valuable alternative to chemical treatment. So successful are some virus pesticides that chemical controls are no longer necessary. The introduction of the myxoma virus into the rabbit popu lation of Australia is a classic example of the effectiveness of this approach (Fenner, 1959). No less dramatic has been the use of viruses in the control of insect past (Rivers, 1964). Virus disease reflects a basic approach to algal control, the effects of which resem ble the fluctuations and succession little understood in the natural envi!onment. In these controls are undoubtedly hidden the clues for efficient treatl1'lent of troublesome cyanobac terial popUlations. Before the discovery of first Cyanophage by Safferman and Morris (1963), Krauss (1961) had predicted their presence in natural ecosytem. His predictio n was based on the sudden coll apse of cyanoba cterial (blue g reen al gal) bl ooms when nutrient level s and environmental con di ti ons were still id eal for continued algal growth. Subsequent to the discovery of the LPP- 1 cyanopha ge by Safferman and Morri ( 1963 ), several other cyanophages and their strains which infec t either unicellu lar or fil a­ mentous non heterocystous or heterocystou s hosts have been described (Ad ol ph and Haselkorn, 1971; Brown, 1972; Hu et 61. 1981 . Khudyakar et al. 1973; Kozyakov et al.,1972;

Fifth Annual Session ]Q86 K~zyakov,

1977; Safferman. 1968; Safferman, 1973; Safferman and Morris, 1967; Saffer­ man et aI., 1969 Suguna. 1980). Several workers in the field of cyanophage research have suggested that the cyanophages playa role in the ecology of blue g reen algae (cyanobacteria). and that they are very possibly effecting natural control of algal blooms in natural water bod:es (Brown, 1972; Cannon, 1976; Granhall, 1972; Krauss, 1961; Lin, 1972; Safferman, 1968; Safferman, 1973; Safferman and Morris, 1964; 1967; Shilo, 1969; 1972) if they were to be introduced

by man.

Saffermnn and Morris (1964) were the first to suggest this poten­

tial. Thev also pointed out that the approach for control of nuisance algal species

permits

their replacement vvith more desirable species without total annihilation of algal popula­ tions which might well occur when chemical algicides are used. Be c ause cyanophages halle, on th~ whole, a rather high host specificity they would appear to be ideal for the selective elirnination of nuisance species. The "idealness" of cyanophages as an algiside has been previously pointed out and discussed (Desjardins et aI., 1978; 1981). The development of nuisance levels of cyanobacterial species in bodies of fresh water is merely a symptom of inr.reased eutrophication of such water bodies which ,"rises from man's distrubance of the ecological balance in nature. It has been noted that cyanobacterial blooms can themselves contribute to the increased eutrophication of water bodies by increasing the level of organic matter when the blooms deca\,. and by the production of extracellular polysaccharides (Barkley and Desjardins, 1976, Berg, 1976). IJIJh8n blue green algal (cyanobacterial) cell densities build up to nuisance levels they must be considered as water poll utant. Some species are toxic to man, domestic animal s an d fish (Lin. 1972; Shilo, 196 7; 1969). Some species also kill aquatic life by oxygen depletion which oc curs duri n g decom position of larg e algal masses (Shilo, 1969). Sometimes very high cell populations, just by their great numbers, interfere with water usage and trearnent. Other species destroy water quality from the sttlnd point of taste and odour. bodies.

The latter situation has at t imes been a serious problem in some water

Control of blue green algal (cyanobacterial) removal or removal by nutrients is an exp3nsiv3 and

blooms either by their physical continuing process.

Chemical

aigicides, in addition to being expensive often have adverse side effects on organisms in the food chain. In addition, the pot en ti al of chemical algicides for disturbing the natural ecology of the water must not be overlooked. There is ample evidence that some clones of certain

species of cyanobacteria

2

M . P. Vigyan Academy ,do indeed produce very potent toxins (Barton and Johnson. 1978; Carmichoel. 1981; Carmichael and Gorharm. 1981; Collins. 1978; Gorham and Carmichael. 1979; Holm­ Hansen 1968; Hughes et aI., 1958; ~oster and Orcutt 1980). The more frequent d etection of blooms of toxic algal species in recent years makes the nuisance aspect of algal blooms even mOre serious. It is obvious of course, that cyanobacterial populations in water bodies need to be managed so that bloom concentrations do not occur. In this address I shall co nsider the possibility of using cyanophages in control and management stratagies. Safferman and Morris (1963) reported the discovery of the first cyanophage. The LPP-1 cyanophage was first described 23 years ago which infects cyanobacteria in the genera Lyngbya. Plw­

rmidiw1l and Plectonama (Safferman and Morris, 1963). In subsequent years additional cyanophages and cyanophage strain, infecting both unicellular, and filameutous cyano­ bacteria have been described (Brown. 1972; Desjardins, 1981; Desjardins and Olson. 1981; Granhall. 1972; Hu et al .. 1981; Khydyako'J. 1977; Safferman. 1973; Safferman et al., 1969; 1972. Safferman and Haselkorn, 1971) viz. LPP-1. LPP·2. A-1 . C-l. N·1, AS-1. SM·1.

PROPERTIES OF CYANOPHAGES Cyanophages are similar in morphology and interaction with their host.> to many of the bacteriophages since the hosts of both groups of phages are both prokaryotes and related. Cyanophages adsorb to their host cells prior to actual infection much the same way that many bacteriophages adsorb to their hosts (Currier anrl Work, 1979; Bisen et al • 1995. 1986). They use the tail structure to inject their infectious nucleic acid into host cells. In the latter respect they also resemble many bacteriophages. In another respect they differ dramatically from bacteriophages and thai is in their requirement for light for adsorp­ tion to their host cells (Allen and Hutchinson . 1976; Csek9 and Far"as. 1979). In the As-1 A . nidu/ans system adsorption varies directly with the intensity of red light. In the dark, adsorption diminishes to almost zero (Allen and Hutchinson. 1976; Cseke and Farkas 1979). All cyanophages that have been characterised to date have doule standed DNA (Brown. 1972; Desjardins. 1981; Safferman. 1973 a. b; Sherman and Brown. 1978). The cyanophages are generally stable over a somewhat larger pH range (pH 4-11) than most bacteriophages (pH 5-8) (Safferman and Morris. 1964. Sherman and Brown,

3

,/

Fifth Annual Session 1986 1978 Bisen and Audholia, 1986).

Their stability and ability to infect their hosts at

alkaline pH's is to their advantage since many cyanobacteria thrive at pH levels of n eutra­ lity or above (Brock . 1973). Some cyanophages are lytic so called because they cause lysis and death of their host cells. The lytic capability of a cyanophage generally speaking, recommended it as a biological control agent. This lytic capability can best be illustrated by the clear plaques they produce on lawns of their host cell.

The clear plaques represe nt areas of the cell

layer (or lawn) where host cells have been lysed by the infecting cyanophage. It has been demonstrated in the laboratory (Bisen et aI, 1985; 1986) that when an algal host is continuously exposed to a specific cyanophage a resistant host strain becomes dominant in the culture. Under the pressure of the lytic phage there is a sele­ ction for resistant strains (Bisen et al., 1985, 1986; Canon et al., 1976; Jenifar, 1977), It has even been suggested that the appearance of these resistant strains in laboratory cultures leduces the prospect of successfully using cyanophages as biological agents (Jenifer, 1977). Such reasoning suggests that one can extrapolate results from relatively simple laboratory experiments where all parc.lmeters of nutrient level and environmental factors can be rigidly controlled to natural ecosystems where there is continual variation in all of these parameters. It has already been pointed out that such reasoning completely ignores the succession of dominant species which occurs in natural water bodies. It has Ellso been shown that even under laboratory conditions a susceptible strain of the host might agilin become the dominant species when the cyanophage is no longer present and its selective p:essure no longer effective (Desjardins 1981). Cannon (1975) has also indica­ :ed that exhaustive fif::ld studies have failed to demonstrate the presence of build up of resistant hot strains in nature in locations where specific cyanophages have been found. The fact that is most natural locations where cyanophages have been found, their hosts are either not found or found in very low cell populations has been interpreted as natural control (Sterens and Sterens, 1980). The Phenomenon of lysogeny in cyanophage-host systems has been established (Bisen et 81.,1986; Hu et cli., 1981; Khydyakov and Gromov, 1973, Kozyakov et al., 1972 Ri:non and Oppenheim, 1975; Sherman and Brown, 1978; Shilo, 1972, Cannon et al. 1971). However, induction of the lytic state from the lysogenic condition appears to be more difficult with cyanophages than bacteriophages (Bifen et al., 1985, 1986; Rimon and Oppenheim, '1975 Sherman and BrOwn, 1978 Shilo, 1972). When the lysogenic condition is established the cyanophage viral genome b9comes integrated with th9 host chromsom9 and does not replicate sepilrately nor cause 4

M. P. Vigyan Academ

lysis of th3 h(}st cell. Many of the viral genes that caie for virion stru ctural protein ~ destruction of host DNA. and lysis of the host cell are not expressed.

Generally within a range of temperature the rates of chemical and biochemic:a reactions increase with increased temperature. Temperature apparenly plays an importan role in the development of natural blooms (Kruger and Eloff, 1978). As one might ex pect temperature also affects the growth cycle of cyanophages (Allen and Hutchinson, 1976 Sherman and Brown 1978). In the AS-1--A. niduians system, the length of the growtl cycle varies inversely with temperature (Olsen and Desjardins 1982). In the range a 25 to 26° C a 1 0 C increase in temperature shortens the growth cycle by approximatel1 0.5 hours.

A truly uniq~e feature of cyanophages in which they differ from their bacteriophag! counterparts is their dependence on the photosynthetic activity of their hosts for thei replication (Adolph and Haselkorn, 1972; Sherman and Haselkorn 1971; Sherman an( Brown 1978). Although the cyanophages that inject unicellular cyanobacteria and thost that infect filamentous cyanobacteria react the same in some respects to any impairmen to the photosynthetic activity of their hosts, in other respects they differ Callen and

Haselkom, 1972, Sherman and Brown,1978).Both groups are dependent on photosystem I and both are inhibited by carbonyl·cyanide m·chlorophenyl hydrazone (CCCP), ar

In h i bitor of fJh otosynthetic electron tra nsport. I n the fi nal ana Iys is both depend on thE availability of ATP. They differ in that unicellular cyanophages do not inhibit CO 2 fixatior while the filame;ttous group does. The latter group is not dependent on photosystem II where as the unicellular group is.

In the As-1-A. niduians system at at 30° C an inverse log linear correlation was found with far red light and the length of the rise period of the growth cycle of the cyanophage. A linear relationship between the red far red ratio and virus yield was also found (Olson and Desjardins 1982). Thus not only does the light quality playa role in the replication of the virus, but the ratio of red to far red is also effective in virus yield. The latter has apparently not been recognised before and is, I believe quite significant. The adsorption

rate of

the N-1 virus

to its host Nostoc niuscO/,1I11l

could be

increased by increasing the host cell density (Adolph and Haselkorn, 1972), but the rate of adsorption of AS-1 virus to A. niduians was not altered by increasing host cell concen­ tration (Ba(kley 1976). According to Adolph and Haselkron (1972) in their work with N-1 virus infe­ ctions of N. I11liSC O rllm, COl fixation is not essential for virus replication; in fact they found

5

Fifch AnnIJal Session 1986 a reduced photoassimilation of CO 2 between 4 to 5 hou rs after infection. They indicated that photosynthesis appeared to be necessary only for the production of ATP. Padan and Shilo (1973) and Bisen et al. (1986) have also pointed out that if an adequate supply of ATP is provided LPP cyanophage can replicate in the dark and un der I. oodit ions which inhibit host growth. One might consider the ionic environment as a factor in the control of their hosts by cyanophages although there is not much experi­ mental data upon which to base on assessment. It has been shown that Mg++ ion is necessary for the stability of LPP-1 virus and for its adsorption to its host (Mendzhul et al. 1974). but the

required

concentrations

are

low, and

therefore,

should not be a limiting factor. One must wonder however, what effect extremely high salt concentration might have on cyanophage host interaction and on cyanophage sta bility. It is conceivable that high salt concentration might affect adsorption of virus partic les to organic debries and also might have an osmotic effect on the virus particle itself Such effects have yet to be investigated. ATTEMPTS AT VIRAL CONTROL OF CYANOBACTERIA

As stated earlier, su~sequent to the first report by Safferman and Morris (1963) of the isolation of the LPP-1 cyanophage several viruses and virus strains which infect unicellular or fil3mentous cyanobacteria have been reported. The emphasis of research has been on t[le purification of these viruses and the characterization of their physical, chemisal and biological properties. Although this emphasis of research efforts is under­ stilndnble because of the novelty of the virus group, it is still somewhat puzzling as to vvhy grea t er efforts have been made to demonstrate their control potential partial expla­ na ion of this lack of effort was due to the realization that certain basic knowledge of the viruses and their properties is essential for the selection of approaches to testing the actual control potentiill. Jackson and Siadecek (1970) made one of the first attempts to actually control blue-green algae in a natural habitat. They attempted to establish P. boryal1um in 5000-9allon tanks at a sewage treatment plant in order to test the LPP-1 virus against this host. Over 40 attempts were made to establish the host in the tanks and all were unsuccessful. The LPP 1 virus was already naturally ~resent and was controlling the algal host and preventing its establishment. It has been reported that Russian workers have controlled blue-green algal scums a result of sprayin!=) cyanophages on infested waters (cited in Deelen, 1969). The cyanophages utilized and their hosts were not described. 65

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M. P. Vigy an Academ

DISCUSSION, RESEARCH NEEDS AND PRIORITIES

In this paper I have attemp.ted to briefly summarize the information on cyano­ phages that is in any way related to their potential as biological control agents. Also have attempted to describe possible problems which should be considered both w4t~ respect to the assessment of algal control potential and to further research needs and priorities. It is my opinion that cyanophages do appear to have biolBgical control poten tial but that certain areas of research should be intensively pursued.

An integlated approach utilizing all available methods of biological control migh prove to be the most fruitful for the desired management and control of nuisance species Methods would include the ecosystem man ipulation (biomanipulation) suggested b Shapiro et al. (1975) the use of bacterial agents as recommended by Burnham (1975) and Burnham et al. t 1976), and the use of single or mUltiple cyonophages. In regard to microbial agents, the Myxococcus species would appear to be especially promising for an integrated approach. Some discussion of toxic blue-green algal species should be included in conside­ ring biological control. The most frequently found toxic blue-green algae are Microcystis acrllginosa, Anabael1G. jios-aquae and Aphanizomenon fiosaquae (H ughes, et 01. , 1958; Collins, 1978; Gorham and Carmichael, 1979) but toxic isolates ~ave also been found in Lyngbya, Schizothrix and Synechococcus (Gorham and Carmichael, 1979). To date cyanophages infecting Microcystis aerugillo.Ja (Safferman et al., 1969; Martin et aI., 1978) and Aphollizomenoll fios -aquae (Gran hall, 1972) have been reported; however the one infecting A. fios-aquae has been lost from culturr.l and is no longer available.

Of considerable interest is the recent report by Porter and Orcutt (1980) that algal toxins reduce the survival capabilities of Daphllia. This finding sugg ests that an integrated approach to biological control might indeed be a prudent one. With respect to the use of cyanophages as biological control agents, I would suggest the following research needs: 1. Search for cyanophages for nuisance species, especially toxic species, for which no viruses are presently known .

2. Determine whether virus-resistant host strains develop under natural conditions in large bodies of water.

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Fifth Annual Session 1986 3. More cri tica lly d etermine the role of lysogeny in the ecology of cyanophage-host interactions and how it might affect control. 4. Accurately determine the effect of environmental factors on the infection and survival capabilities of cyanophages. 5. Establish outdoor research facilities which wo uld permit standardization of conditions, as far as possible, between control and cyanophage-treated waters.

6. Initiate tests utilizing presently available cyanophages in natural water bodies where their hosts have been a continuing nuisance.

7. Attempt to elucidate how toxic algal species arise-do plasmids or viruses possibly playa role?

REFERENCES

Adolph, K. W. and R. HaselKorn (1971). : Virology 46: 200-208 Ado!ph, K. W. and R. Haselkorn (1972).: Viroiogy 47 : 370-374. Allen, M. M. and F. Hutchison (1976): Arch. Microbiol110: 55-60 Barkiey, M. B. (1976). : Ph. D. thesis, Univ. Calif., Riverside pp 144 Barkley, M. Band P. R. Desjardins (1977) : App\. and Environ. Microbiol 33: 971-974 Barton, L. L. and G. V. Johnson (1978).: Tech. Completion Report Berg, G. (1976). : J. Water Pollution Control Federation 48 : 1410-1416 Bisen, P. S.. Surabhi Audholia, and A. K. Bhatnagar (1985). : Microbios Letters 28: 7-13 Bisen, P. S., S. N. Bagr.hi and Surabhi Audholia (1986).: FEM S Microbiology Letters 33: 69-72. Bisen, P. S .. Surabhi Audholia, A. K. Bhatnagar and S N. Bag chi (1 986) . : Current Microbiol13: 1·5 Bisen, P. S., Surabhi Audholia and S. N. Bagchi (1986). : J. Plcnt Physio!. (Communicated) 8

M.P. Vigyan Academy Bisen, P. S., and Surabhi Audholia (1986). : Microbios Letters (Communicated) Brown, R. M. Jr. (1972) : Adv. Vir~s. Res . 17 : 243-277. Brown R. M. Jr. (1978).: Compreh. Viro!. 12: 145-234 Bro ck, T. D. (1973). : Science 179 : 480-482 Connon, R. E., M. S. Shane and V. N. Bush (1971). : Virology 45: 149-153 Cannon, R. (1975). : Rept. Proc. Symp. Water Quality Management through biological control Cannon, R. E., M. S. Shane and J. M. Whitaker (1976) : Jour. Phyco!. 12: 418-421 Carmicha el, W. W. (1981) : In the Water Environment Algal Toxins and Health. Plenum Press, New York Carmichael, W. W. and P. R. Gorham (1981). : In the Water Environment Algal Toxins and Health, Plenum Press, New York Collins, M (1978). : Microbiol Rev. 42 : 725-746 Cseke, Cs and G . L. Farkas (1979). : J. Bact. 137 : 667-669 Currier,

1. C. an d C. P. Wolk (1979) . : J our. Bact. 139 : 88-92

Desjardins, P. R. , M. B. Barkley, S. A. Swiec ki and S. N. West (1978).: Califomia Water Resources Center, Univ. Calif. Desjardins p, R. (1981), : Proc. Workshop Algal Manage Control Tech . Rep. Fanner, F. (1959). Brit. Med. Bull. 15 : 140-255 Gorham, P R. and W. W. Carm ich ael (1979). : Pure Appl. Chem . 52: 165-174 Granhal\, U· (1972). : Physiol Plant 26 : 332-337 Hu, N- T, T. Thiel, T. H. Giddings Jr. and C. P. Walk (1981) : Virology 114 : 236-246 Hughes, E. D" P. D. Gorham and A. Zehnder (1958). : Can. J. lVl icro b io l 4: 225-236 Ho lm-Han sen , O. (1968). : Ann. Rev . Microbiol 22: 47-70 J ackson, D. and V. Siad ecek . ; Yale Sci . M ag. 44 : 16-21 Jenifer, F. G. (1977). : Cornple Rep . W ater Reso ur Res. Inst. Ne w Brun swick , Nj.

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Fifth Annual Session 1986 Khydyakov, J. Ya and M V. Gromov (1973). : Mikrobiologiya 42 : 904-907 Kozyakov, S. Y., B.V. Gromov. and I. Y. Khudyakov (1972) : Microbiologia 41 : 486-489 I(ozyakov, S.Y. (1977). : Biological Scfentific Res. Inst. Bull. Leningrad State University. Krauss, R.W. (1961). : In Algae and Metropolitan Wastes U. S. Dept. Health Edn. Welfare SEC TRW 61-63 Lin , C K. (1972) : Hydrobiologia 39: 321· 334 Mendzhul. M .I., S. P. Bobrovnik. and T.G. Lysenko (1974). : Voprosy Virusologii 1 : 31-36 Olson, G B. and P.R . Desjardins (1982), : Abstr. PP 48 XIII Inti. Congress of Microbiology Boston. USA Porter, K. G. and J. D. Orcutt Jr. (1980). : In A. S. L. 0 Special Symp. Iii: The Evolution and Ecology of Zooplankton Communities Rlmon, A . and A. B. Opperiheim (1975) . : Virology 64: 454-463 Rive rs, C. (1964). : Discovery 25 : 27-31 . ~affermen,

SaffermE·n

R.S (1968).: Algae. Man and Environment. Syracuse Univ. Press R. S, (1973).: In J. R. Stein (Ed.). Handbook of Phycological Methods­ Culture methods and growth measurements Cambridge University Press

Safferman. R.S. (1973). : The biology of blue-green algae, Blackwell Sci. Pub. Safferman, R. S. and M.E. Morris (1963)': Science 140: 679-680 Safferman , R. S. and M. E. Morris (1964). : Jour. Amer. Water Works Assoc. 56: 1217-1224 Safferman , R. S. and M.E. Morris (1967) : Appl. Microbiol15: 1219-1222 Safferman, R. S., I. R. Schneider. R. L Steare, M. E. Morris and T.O. Diener (1969) . : Virology 37 : 386-395 Safferman, R. S .. T. O. Diener, P. R. Desjardins and M. E. Morris l1972).: Virology 47 : 105-113 Sherman , L A. and R. Haselkorn (1971). : Virology 45: 739-746 Shiio , M. (1967). : Bact. Rev. 31 : 180-193 Shilo , M. (1969) : Proc lind IntI. Cant. on Globallmpaet of App! ied Microbiology,177-184 Shilo, M. (1972). : Bamidgeh 24 : 76-82 Ste vens, C.L.R. and S. E. Stevens Jr. (1980).: In E. Gantt (Ed .) Handbook of Phycological Methods Developmental and Cytological methods Cambridge Univ. Press Suguna, D.L. (1980) . : Ph. D. Thesis, Madurai Univ ., Madurai. Ind ia.

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