After screening of several rhizosphere bacteria against the soilborne pathogens of

red core and crown rot disease of strawberry Phytophthora fragariae var. fragariae

and Phytophthora cactorum under in vitro conditions, three of the most active isolates

which produced up to 63% of reduction in mycelium growth, such as Raoultella

terrigena (G-584), Bacillus amyloliquefaciens (G-V1) and Pseudomonas fluorescens

(2R1-7) were selected for further studies under in vivo conditions. In a greenhouse

and field experiments, mentioned above three isolates were tested against both

Phytophthora diseases under artificial infested soil conditions. Root dip treatment

with these bacterial antagonists produced a control effect on both fungal diseases

between 27 to 55 % and were in some cases comparable with the chemical fungicide

Aliette

Anandhakumar, J.

Zeller, W.

English

Organic Eprints

Federal  Biological  Research  Centre  for  Agriculture  and  Forestry,  Institute  for  Biological  Control, Heinrichstr.243, 64287 Darmstadt, Germany   Investigation on the Biocontrol of Phytophthora diseases on strawberry based on antagonism  J. Anandhakumar  & W. Zeller   Abstract After screening of  several rhizosphere bacteria against the soilborne pathogens of  red core and crown rot disease of strawberry Phytophthora fragariae var. fragariae and Phytophthora cactorum under in vitro conditions, three of the most active isolates which  produced  up  to  63%  of  reduction  in  mycelium  growth,  such  as  Raoultella terrigena (G-584), Bacillus amyloliquefaciens (G-V1) and Pseudomonas fluorescens (2R1-7) were selected for further studies under in vivo conditions. In a greenhouse  and  field  experiments,  mentioned  above  three  isolates  were  tested  against  both Phytophthora  diseases  under  artificial  infested  soil  conditions.  Root  dip  treatment with these bacterial antagonists produced a control effect on both fungal diseases between 27 to 55 % and were in some cases comparable with the chemical fungicide Aliette.   Key  words:  Biocontrol,  Rhizobacteria,  Phytophthora  fragariae  var.  fragariae, Phytophthora cactorum   Introduction  Strawberry is one of the important berry cultures in Germany, which is attacked by several  viral, bacterial and fungal diseases. Of the fungi attacking strawberries two of the  most  important  pathogens  Phytophthora  fragariae  var.  fragariae  and Phytophthora cactorum, which are the cause of red core and crown rot disease of strawberry,  can  cause  substantial  economical  damage  in  strawberry  production (Seemüller 1998). In areas, where P. fragariae var. fragariae and P. cactorum are present,  cultivation  of  strawberry  depends  directly  on  the  use  of  chemicals.  The number  of  chemical  fungicides  available  for  the  control  of  both  Phytophthora pathogens are limited. In most countries two different fungicides viz. Ridomil  with Metalaxyl  and  Aliette  with  Aluminium-Fosetyl  as  active  substance  are  available against  these  pathogens.  In  Germany,  Aliette  is  the  only  fungicide  registered  for control of red stele disease of strawberry (Anonymous,1999). In some fields where Metalaxyl was used for years, resistant strains of P. fragariae var. fragariae have been found (Nickerson,1998), resistance of Phytophthora species to Metalaxyl has been  documented  by  Seemüller  et  al  (1989).  The  use  of  chemicals  especially compounds  containing  mercury  and  copper  were  widely  resorted  to  in  order  to achieve high levels of disease suppression. However, the persistent, injudicious use of  chemicals  has  been  discouraged  owing  to  their  toxic  effects  on  nontarget organisms and due to the undesirable changes they inflict upon the environment. Many of the chemicals are also too expensive for the resource-poor farmers of Asia (Vasudevan  et al.,2002).  The problems associated  with  the use of  agrochemicals have promoted research in the field of biological control of  plant diseases as an alternative method of control also in the frame of ecological organic farming . Hence in this study, an attempt has been aimed to control both Phytophthora diseases by using different antagonistic bacteria Archived at http://orgprints.org/14548/Federal  Biological  Research  Centre  for  Agriculture  and  Forestry,  Institute  for  Biological  Control, Heinrichstr.243, 64287 Darmstadt, Germany      Material and Methods  Preparation of Bacteria After a screening of more than 100 bacterial isolates out of the rhizosphere of fruit  orchards in dual culture test, three with the best  inhibition effect against the two Phytophthora pathogens, were further used for the in vivo experiments. The following bacterial isolates were used: Raoultella terrigena (G-584), Bacillus amyloliquefaciens (G-V1) and Pseudomonas fluorescens (2R1-7). Bacterial isolates G-V1, G-584 were formulated by the company E-nema, Raisdorf. The 2R1-7 isolate were prepared by growing in Tryptic Soya Broth for 24 h at 26°  C, cells were harvested by centrifugation and diluted to an optical density of 0.20 at 660 nm. The bacterial concentration 108 to 109 used for the experiments.  Preparation of Pathogen Inoculum Inocula  of  P.  fragariae  and  P.  cactroum  were  cultured  in  vermiculate  media (composed of seeds of rye) for 4 weeks at 18°  C and  3 % of inoculum used for artificial infection of soil.  Plant material Tests were performed with strawberry (Fragaria X ananassa Duchesne) cv, " Elsanta " plants. This variety of strawberry is highly susceptibility to red stele and crown rot disease.  Greenhouse Experiments Strawberry plant roots were dipped for 15 minutes into the bacterial suspension in order  to  have  direct  exposure  of  the  roots  to  the  antagonistic  bacteria.  Roots  of control  plants  were  either  dipped  in tap  water  or  0.5  % Aliette.  Plants  were  then transferred to 14 cm plastic pots with artificially infested soil using 3 % of pathogens. The  experiment  was  arranged  in  two  separate  blocks,  each  with  10  pots  per treatment. 90 days of after  planting, plants were removed from pots, the roots were carefully  washed  and  disease  was  scored  using  a  0-5  scale  and  disease  index calculated. Statistical significance was tested using Duncan's Multiple Range Test (P=0.05).  Field Experiments Field experiments were carried out under artificially infested soil conditions (3 % of pathogen  inoculum  was  applied  to  each  planting  hole).  Plants  were  treated  with bacteria by root dip as described above. Plants were planted in four rows at least 40 plants  per  treatment  (arranged  in  randomised  design).  Harvesting  of  plants  (12 weeks after planting), rating  of disease severity, computerization of disease index and statistical significance were all made as described above. Results and Discussion  In greenhouse experiment,  rhizobacteria showed different levels of biocontrol activity towards both pathogens (Table .1). maximum % control of 54% against red core from Archived at http://orgprints.org/14548/Federal  Biological  Research  Centre  for  Agriculture  and  Forestry,  Institute  for  Biological  Control, Heinrichstr.243, 64287 Darmstadt, Germany   isolate  2  R1-7  of  P.  flourescens,  where  as  against  crown  rot  isolate  G-584  of Raoultella  terrigena  showed  the  highest  effect  of  55  %  which  induced  nearly comparable with chemical control Aliette 64. 7 %.  Tab.1:  Influence  of  Rhizobacteria  on  disease  index  of  red  core  and  crown  rot  of strawberry under artificial infested soil conditions in the greenhouse ( 90 days after treatment) n=20                 Red core                  Crown rot  Treatments % Disease index  % Control   % Disease index  % Control Control  42 a    51 a   G-584  26 bc  38.1  23 b  55.0 G-V1  27 bc  35.7  26 b  49.0 2 R1-7  19 c  54.7  26 b  49.0 Aliette  16 c  61.9  18 b  64.7  The tested  bacterial isolates also gave a considerable degree of protection against both pathogens. In a field experiment (Table. 2), the isolate 2 R1-7 of P. fluorescens showed also highest effect up to 50% followed by G-584 and G-V1 against red core disease but a lower degree of control against crwon rot (32 to 37 %).   Bacterial  strains  tested  for  biocontrol  activity  towards  Phytophthora  sps  were previously shown to have biocontrol activity against different pathogens on a variety of different crops except bacterial isolate Raoultella terrigena of our knowledge. In our studies, all of the tested strains showed different level of protection against both Phytophthora sps, variation in antagonistic activity of rhizobacteria with time and in different systems is a known phenomenon (Weller 1988) and still difficult to explain. Biotic factors such as concurrence in root colonization with the soil specific microbial community or infection by pathogens may play a role. Abiotic factors such as soil type, moisture and temperature as well as variations in application technology also may of importance (Deacon 1991).  In addition, isolation and identification of  Phytophthora spp from naturally infested soil from fields of different growers were undertaken, in order to confirm the natural infested soil status for field experiments. Isolates were identified at the genus level by using  selective  media,  microscopic  observation  and    plant  disease  detection  kit (ALERT, Neogen corporation ,USA).  Further biocontrol studies are in progress on different locations of Germany under naturally  infested  soil  conditions  with  strawberry  farmers  in  order  to  confirm  the efficacy. Investigations on the bacterial mode of action of their biocontrol activity will help in understanding  the differences in activity between the bacterial strains.    Archived at http://orgprints.org/14548/Federal  Biological  Research  Centre  for  Agriculture  and  Forestry,  Institute  for  Biological  Control, Heinrichstr.243, 64287 Darmstadt, Germany   Table. 2. Influence of Rhizobacteria on disease index of red core and crown rot of strawberry under artificial infested soil conditions in the field (June 2003) ( 90 days after treatment) n=40                  Red core                  Crown rot  Treatments % Disease index  % Control   % Disease index  % Control Infected Control 37.5 a    29.0 a   G-584  23.0 b  38.6  18.0 bc  37.9 G-V1  22.5 b  40.0  19.5 bc   32.7 2 R1-7  18.5 b  50.6  19.0 bc  34.4 Aliette  18.0 b  52.0  14.5 c  50.0 Untreated control 6.5 c    7.0 d    The studies have been undertaken in cooperation with firm E-Nema, Raisdof with the aim to develop  an antagonistic preparation against both Phytophthora diseases.  Reference  1.  Anonymous (1999) Pflanzenschutzmittel-Verzeichnis Teil 2 1999 Gemüsebau-Obstbau-Zierpflanzenbau 47. Auflage 1999, ISSN 0178-0603 Biologische Bundesanstalt für Land-und Forstwirtschaft Bundesrepublik Deutshland. 2.  Decon, J. W.: Significance of ecology in the development of biocontrol agents against soil-borne plant pathogens- Biocontrol Sci.Technol. 1, 5-20, 1991. 3.  Nickerson, N. L. (1998) Red stele root rot, In Compendium of Strawberry Diseases, Second Edition. Maas, J. L. (ed.). APS Press 48-50. 4.  Seemüller, E. (1998) Crown rot, In Compendium of Strawberry Diseases, Second Edition. Maas, J. L. (ed.). APS Press 50-51. 5.  Seemuller, E. and  Sun, C. (1989) Auftreten von Metalaxyl-Resistenz bei Phytophthora fragariae. Nachrichtenblatt Deutsche Pflanzenschutzdienst. 41: 71-73. 6.  Weller, D. M.: Biological control of soilborne plant pathogens in the rhizosphere with bacteria- Ann. Rev. Phytopathol. 26, 379-407, 1998. 7.  Preeti Vasudevan, S. Kavitha, V. Brinda Priyadarishini, Lavanda Babujee, and Samuel S. Gnanamanickam : Biological Control of Rice Diseases- Biological control of Crop Diseases edited by Samuel S. Gnanamanickam, 2002. Archived at http://orgprints.org/14548/

Investigation on the Biocontrol of Phytophthora diseases on strawberry based on antagonism

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