Inoculum density of Plectosporium alismatis, a potential mycoherbicide, in relation to control of the aquatic weed Sagittaria montevidensis

Densidade de inóculo de Plectosporium alismatis, um potencial micoherbicida, em relação ao controle de Sagittaria montevidensis

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Inoculum density of Plectosporium alismatis, a potential mycoherbicide, in relation to control of the aquatic weed Sagittaria montevidensis

Densidade de inóculo de Plectosporium alismatis, um potencial micoherbicida, em relação ao controle de Sagittaria montevidensis

Brenda Ventura Lima; Dartanhã J. Soares^*; Robert W. Barreto

Departamento de Fitopatologia, Universidade Federal de Viçosa, 36570-000, Viçosa, MG, Brazil

ABSTRACT

Sagitaria montevidensis (arrowhead) is one of the worst weeds of irrigated rice. Control of this weed by means of chemical herbicides has become increasingly difficult on account of herbicide resistance in populations of S. montevidensis. Mycoherbicides are recognized as potential alternative tools against S. montevidensis. In Australia, the fungus Plectosporium alismatis was reported as a possible mycoherbicide for Damansonium minus and other alismataceous weeds, but Australian isolates of the pathogen did not infect S. montevidensis. In contrast, Brazilian isolates of P. alismatis obtained from S. montevidensis have shown promise in controlling the weed. The present study investigated effects of different inoculum densities of a selected strain of P. alismatis on S. montevidensis. Respective densities of 2 x 10⁶ and 2 x 10⁷ conidia.mL^-1 caused an average of 86 and 93 % leaf blight followed by growth cessation and death of the plants. It was concluded that a density of 2 x 10⁶ conidia.mL^-1 was sufficient to control S. montevidensis and that this density should be adopted as standard in future experiments.

Key words: Rhynchosporium alismatis, arrowhead, biological control, mycoherbicide.

RESUMO

Sagitaria montevidensis (sagitária ou aguapé-flecha) é uma das plantas invasoras mais nocivas na cultura do arroz irrigado. O controle desta invasora tem se tornado difícil devido ao surgimento de populações desta espécie resistentes a herbicidas. O uso de micoherbicidas pode vir a ser uma alternativa para contornar este problema. Na Austrália o fungo Plectosporium alismatis já foi detalhadamente investigado como potencial micoherbicida para Damansonium minus e outras espécies da família Alismataceae, entretanto os isolados australianos deste patógeno não eram infectivos a S. montevidensis. Isolados deste fungo obtidos no Brasil foram promissores no controle de S. montevidensis. O presente estudo investigou o efeito de diferentes densidades de inóculo de P. alismatis para o controle de S. montevidensis. Densidades de 2 x 10⁶ e 2 x 10⁷ conídios.mL^-1 causaram em media 86 e 93 % de queima foliar seguido de paralisação do crescimento e morte das plantas. Concluiu-se que uma densidade de inóculo de 2 x 10⁶ conídios.mL^-1 seria suficiente para o controle de S. montevidensis e que esta densidade deveria ser adotada como padrão em futuros experimentos.

Palavras-chave: Rhynchosporium alismatis, aguapé-flecha, controle biológico, micoherbicida.

Sagittaria montevidensis Cham. & Schltdl. is a South American aquatic plant of the Alismataceae (Lorenzi, 2000); it is considered one of the worst weeds in irrigated rice in the southern Brazilian states of Rio Grande do Sul and Santa Catarina. It is also an important weed in Australia and the USA. ALS-synthesis inhibiting herbicides are widely used for controlling the weed, but herbicide-resistant populations of S. montevidensis are increasing worldwide, making chemical control difficult (Pratley et al., 2001; Concenço et al., 2007). In recent years biological control has gained increasing attention as an option for controlling alismataceous weeds, including S. montevidensis. Soares et al. (2009) surveyed fungal pathogens of S. montevidensis in Brazil, in an effort to identify potential biocontrol agents. Based on field observations, pathogenicity tests, and other available information, Plectosporium alismatis (Oudem.) W.M. Pitt, W. Gams & U. Braun was selected for studies as a potential mycoherbicide of S. montevidensis.

Plectosporium alismatis is an anamorphic hyphomycete with hyaline, straight to slightly navicular, two-celled phialoconidia, which has been recorded on several alismataceous hosts (Pitt & Gams, 2005; Soares et al., 2009). This pathogen was investigated extensively in Australia for controlling Damansonium minus, Alisma lanceolatum and other alismataceous weeds in irrigated rice (Cother & Gilbert, 1994a, 1994b; Cother & Van de Ven, 1999; Lanoiselet et al., 2001; Jahromi et al., 2002, 2004, 2006; Cliquet et al., 2004; Pitt et al., 2004a; Ghajar et al., 2006; Cliquet & Zeeshan, 2008).

Until recently, P. alismatis was regarded as not being capable of infecting S. montevidensis (Ash et al., 2008; Pitt et al., 2004b); however, Soares et al. (2009) reported P. alismatis infections on S. montevidensis as being rather common in Brazil. Earlier abstracts published in conference proceedings by R. A. Pitelli and co-workers referred to a fungus attacking S. montevidensis as Cylindrocarpon sp. and recognized it as having potential for development as a mycoherbicide. From our experience, it is considered likely that fungi isolated from S. montevidensis and reported as Cylindrocarpon sp. were misidentified and were probably isolates of P. alismatis. Re-examination of these isolates appears justified, especially given the similar morphology of the two genera. Soares et al. (2009) considered that a fungus described in Japan as Cylindrocarpon sagittariae Negeshi was also misidentified.

Commercial development of a fungus as a mycoherbicide generally requires that inoculum can be mass-produced at low cost under controlled conditions. A preliminary assay was conducted involving 14 liquid media recipes (unpublished data), aimed at finding a medium that might be adequate for mass-producing P. alismatis conidia and that meets with the requirements of commercial production. The media included lima bean broth (LBB) as described by Cother and Van de Ven (1999) and thirteen others. Since soybean flour (SF) medium (Vieira, 2008) yielded abundant infective conidia (an average of 10⁷ conidia.mL^-1), and is of simple composition it was chosen for inoculum production in the present work.

The aim of the present work was to determine the effectiveness of P. alismatis against S. montevidensis and the minimum inoculum density of the pathogen needed to provoke disease that is sufficiently severe to reduce or stop growth of S. montevidensis. The findings would provide a basis to further evaluate the potential of Brazilian strains of the pathogen for use in arrowhead biocontrol.

Among four available isolates of P. alismatis (DJS-163b; DJS-166b; DJS-458b and RWB-814a) RWB 814a was the most virulent on S. montevidensis (unpublished data) and was selected for use in a bioassay. Plants of S. montevidensis used in the assay were grown from seeds collected from plants at the Lagoa da Pampulha (Belo Horizonte, state of Minas Gerais). The seeds were sown in pots containing water-saturated soil. The pots were positioned inside 500 L water tanks such that the top of each pot was 10 cm deep in the water. Each plant had at least four fully-developed sagitate leaves above the water. The tanks were located outdoors at an experimental field on the campus of the Universidade Federal de Viçosa and the experiment was conducted during late winter and early spring (August-September) 2008.

Inoculum of P. alismatis for use in the bioassay was produced as follows: 100 mL of SF was dispensed into each of several 250 mL Erlenmeyer flasks, autoclaved at 121ºC for 10 min, allowed to cool, and seeded with discs from 7-day colonies of isolate RWB-814a grown on plates containing Vegetal Broth Agar (Pereira et al., 2003). The flasks were placed on a controlled temperature orbital shaker operated at 150 rpm and maintained with 12 h light/dark cycle at 25 ± 2ºC for 7 days. The colonized liquid medium was filtered through muslin to remove the mycelial mass, and the filtrate containing the conidia and residues of the SF medium was diluted with sterile distilled water to provide inoculum densities of 2 x 10³, 2 x 10⁴, 2 x 10⁵, 2 x 10⁶ and 2 x 10⁷ conidia.mL^-1. The initial conidial density in the filtrate was estimated by means of haemocytometer counts.

Healthy leaves of S. montevidensis plants in the bioassay were spray-inoculated to run-off with the conidial suspensions. The inoculations were performed late in the afternoon so that no special care for maintaining high humidity levels for inoculated plants was necessary. Inoculated plants were observed daily until the first symptoms appeared.

The experimental design was completely randomized with three replicate plants per treatment. Control plants were not inoculated. Seven days after the inoculations, the largest leaf of each plant was detached and its petiole was removed. Each detached leaf was separately and immediately scanned using an HP Scanjet 6100c scanner (Hewlett Packard Company). The plants, each with at least three remaining inoculated leaves, were left in the water tanks to allow further disease development. Scanned images of the leaves were assessed with the software Quant^® (Vale et al., 2004) to determine the percent leaf area that was healthy and the percent area with disease symptoms. The data were submitted to homocedastic test and analysis of variance (ANOVA) using SAS^® (version 9.1). The equation and curve adjusted to the data were obtained using a version trial of the Table Curve 2D^® v5.01 available at SYSTAT Software Inc. homepage (http://www.systat.com).

The evaluations of the proportion of diseased leaf area, as provided by use of Quant^® software, are exemplified in Figure 1. There was a high correlation between the spore density and necrotic leaf area (Figure 2). In this assay the best inoculum density was considered as the lowest density that was sufficient to result in > 80% leaf area blighted.

Moderate to severe symptoms appeared at three to four days after inoculations in leaves inoculated with a high density of inoculum (10⁶ and 10⁷ conidia.mL^-1). Conidial densities of 10⁶ and 10⁷ conidia.mL^-1 resulted in severe leaf blight (average 86.1 and 93.3 %, respectively), interruption of plant growth and subsequently (7 to 20 days after application) death of the foliage, including the leaf blades and petioles. At the high inoculum densities, new leaves that emerged up to 10 days after inoculation also died. Mean disease severity on detached leaves inoculated respectively with densities of 10³, 10⁴ or 10⁵ conidia.mL^-1 was 0.85, 17.6 and 57.7%. At these lower densities the inoculated plants maintained growth, produced new leaves and, after about 30 days, also flowered.

We conclude that an inoculum density of 2 x 10⁶ conidia.mL^-1 is adequate for application of P. alismatis aimed at controlling S. montevidensis and suggest that this density be used in further studies towards the development of a mycoherbicide for control of S. montevidensis. A similar level of inoculum density (10⁶ conidia.mL^-1) was found by Cother and Gilbert (1993) and Jahromi et al. (2004) to be adequate for controlling D. minus and other alismataceous weeds. We have also demonstrated that S. montevidensis is a host of P. alismatis and that, in contrast to findings of Pitt et al. (2004b) for Australian isolates, that Brazilian isolates of P. alismatis are pathogenic to arrowhead. Our findings underscore the opportunity to exploit P. alismatis for development as a mycoherbicide against S. montevidensis.

ACKNOWLEDGMENTS

The authors wish to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (Proc. 482720/2007-7) and Fundação de Amparo à Pesquisa de Minas Gerais - FAPEMIG (Proc. CRA 1786/05) for financial support. D.J. Soares thanks Dr. Eric Cother and Dr. Sophie Cliquet for their help in a literature search and Wirton M. Coutinho for help in the statistical analyses.

Received 11 January 2010

Accepted 21 August 2010

Author for correspondence: Dartanhã J. Soares, e-mail: [e-mail]

TPP 9158

Section Editor: John C. Sutton

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*

Present Address: Embrapa Algodão, 58428-095, Campina Grande, PB, Brazil.

Publication Dates

• Publication in this collection
  03 Nov 2010
• Date of issue
  Aug 2010

History

• Received
  11 Jan 2010
• Accepted
  21 Aug 2010