Assessment of Fungal Pathogens Affecting the Weed Conyza bonariensis in Argentina

In the last years Conyza bonariensis has become an important weed and control is difficult with the use of current technology in Argentinean pampas region. The increasing prevalence of herbicide-resistant weed species, public concern related to pesticide use and the introduction of government policies for pesticide reduction, is driving the search for alternative methods to chemical control. The aims of the present study were to detect fungal diseases associated with C. bonariensis, to identify fungal isolates from the symptomatic leaves and to confirm through Koch’s postulates the isolates pathogenicity. Mycological analysis of symptomatic leaves showed the presence of twelve genera of filamentous fungi. Among 116 isolates, Colletotrichum spp. was the most prevalent genus followed by Nigrospora spp. and Septoria spp. In the pathogenicity assays, 22 out of 116 isolates were able to comply with the Koch’s postulates. The pathogenic isolates were included into three genera Alternaria spp., Colletotrichum spp. and Septoria spp. This study provides the first report that demonstrates pathogenicity of fungal isolates on C. bonariensis in Argentina and represents the first step in a future biocontrol program.


Introduction
Over the past 25 years Argentina has adopted a productive model based on no-tillage cultivation with scarce or no crops rotation, herbicide overuse with glyphosate predominance and mainly lack of awareness, which has led to weed shift towards difficult to control species and evolution of herbicides resistance (Papa & Tuesca, 2014a).Thus, the number of herbicide-resistant weeds increased markedly in recent years in Argentina, with a rate of 4 biotypes per year with a total of 30 resistant biotypes already confirmed (Acciaresi et al., 2017).control.It is crucial to apply herbicides when the plant is a small rosette, preferably of 5 cm in diameter or smaller, and definitely prior to stem elongation until 15 cm, as control efficacy declined as plants mature (Metzler, Puricelli, & Papa, 2013;Papa, Tuesca, & Nisensohn, 2010).However, under the current production model more than 60% of the agricultural area in Argentina is under rental with very short term contracts.As a result there is a late start of activities and weeds control is often carried out late to be treated effectively with normal doses of herbicides available.Conyza bonariensis is characterized as extremely aggressive weed in soybean and can cause 50% or more yield losses if left uncontrolled (Papa & Tuesca, 2014b).Integrated Weed Management (IWM) is defined as the integration of different control methods to provide the crop with an advantage over the weeds (Lamichhane et al., 2017).It is practiced worldwide with different adoption levels and is intended to restrict weed populations to manageable levels, to reduce the environmental impact of individual practices, to increase the sustainability of cropping systems and to reduce selection pressure for weed resistance to herbicides.However, published articles on chemical control since 1995 are the most numerous compared to any other method of weed control (Harker & O'Donovan, 2013).
Considering the general trend to reduce the entry of synthetic pesticides in the agroecosystem, it is necessary to search biological alternatives for example through the use of bioherbicides considered to be a type of inundative biological control.This biological weed control is relevant to the needs of agriculture and turf management, as it can be implemented through the application of inoculum as liquid sprays or solid granules in a similar way to conventional herbicides (Auld, Hetherington, & Smith, 2003).On the other hand, phytopathogenic fungi can be used as producers of bioactive molecules with herbicidal activity (Castro de Souza et al., 2017).Many steps are involved in the implementation of a biological control program but it always includes surveys aimed at identifying the natural enemies associated with the plant in its centre of origin and often also comprises surveys in the region where introductions will be made (Duarte, Santos, & Barreto, 2016).However, no systematic surveys have been carried out on C. bonariensis for suitable agents as it was suggested by Scott et al. (2016).Based on this context, the aims of the present study were: i) to detect fungal disease associated to C. bonariensis; ii) to identify fungal isolates from the symptomatic leaves; iii) to confirm through Koch's postulates the isolates pathogenicity.

Sampling Sites and Leaves Symptoms Analysis
Samples were collected during the periods 2015 and 2016 growing season in experimental fields at the Universidad Nacional de Río Cuarto (33°06′31.6″S; 64°18′01.3″W) (Río Cuarto, Córdoba).Within this area survey sites were arbitrarily selected according the occurrence of weed populations in the production crops, pastures areas, etc. Whenever diseased plants were found, representative parts bearing symptoms were taken and referenced.In the laboratory, infected plant tissues were observed under a magnifying glass (Motic, model DM-39-N9GO) to observe signs, i.e. the presence of fungal structures such as fruiting bodies, conidiophores, sclerotia, etc., establishing associations between signs and disease symptoms.Symptoms on leaves were classified as: central, apical or lateral blight and/or leaf spots.Symptoms were rated using a 0-4 scale according to the approximate leaf blight severity area: 0 = 0%, 1 = 1-10%, 2 = 11-25%, 3 = 26-40%, 4 ≥ 40% (Figure 1).To calculate the percentage of affected leaf an ImageJ 1.51 program was used (Schneider, Rasband, & Eliceiri, 2012).

Funga
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Results
In with ontrol zer of lants, vegetables, ornamental plants and fruit trees as citric and apple tree (Agrios, 2005).Previous studies demonstrated saprophytic associations between Alternaria and Conyza species (Caretta et al., 1999;Roy et al., 1994) or related the fungal presence to lesion on leaves (Duarte et al., 2016).In this study, 2 out 10 Alternaria isolates were able to develop symptoms in C. bonariensis and were subsequently isolated from all lesions.Until now, there is only one worldwide registered bioherbicide called Smoulder® based on A. destruens L. Simmons strain 059.This fungus is parasitic on cuscuta's species and can be apply in crops as alfalfa, carrot, tomato and ornamental plants in USA (Bailey, 2014).
The Colletotrichum genus was the most frequently isolated from symptomatic leaves of C. bonariensis.Similar results were obtained by Roy et al. (1994), andFormento (2013), who demonstrated the presence of Colletotrichum or Glomerella isolates from foliar lesions of Conyza spp.The lesions were similar to those observed in Alternaria isolates; however in the pathogenicity tests a 37% of the isolates were able to develop symptoms and could be recovered from affected tissues.Numerous researches showed association between Colletotrichum species and weed biocontrol and some of them have been registered such as Collego®/ LockDown®, C. gloeosporioides f. sp.aeschynomene ATCC 20358 utilized to control Aeschynomene virginica in rice in USA (Bowers, 1986) and Biomal®, Colletotrichum gloeosporioides f. sp.malvae ATCC 20767 used to control broadleaf weeds in the family Malvaceae Malva in Canadá (Boyetchko, Bailey, Hynes, & Peng, 2007).
The third genus that demonstrated pathogenicity in C. bonariensis was represented by isolates belonging to Septoria spp.In this case, all isolates recovered from symptomatic leaves reproduced the disease in pathogenicity tests showing similar aggressiveness.Previous works worldwide have detected diseases in Conyza related to the presence of Septoria species (Duarte et al., 2016;Erper et al., 2010;Priest, 2006;Fatehi et al., 1993).On the other hand, several investigations have reported the use of different Septoria species as biocontrol agents such as S. cirsii in Cirsium arvense, a weed of Asteraceas's family (Leth, 1985), S. polygonorum proposed against species of Polygonum (Mitchell, 2003) and S. urticae proposed against Urtica urens L. (Bello, Perelló, & Monaco, 1993).
Taking into account that isolates from three genera pathogenic to C. bonariensis have been used as bioherbicides worldwide, such strains could be considered potential candidates as weed biocontrol agents.On the other hand, we know that some Septoria species and others belonging to the Colletotrichum species complex include pathogens in several crops such as soyabean, maize, wheat, sorghum and grasses (Agrios, 2005).These pathogens can survive in the host absence through the use of alternative hosts as weeds or living as saprophytic on crop residues.For this reason, before including a strain into a biocontrol program of C. bonariensis it is necessary to evaluate the environmental risk of the potential bioherbicide using scientific criteria through the evaluation of host specificity, crop tolerance, environmental fate and toxicology.Changes in the public's attitude on the acceptance of synthetic herbicides (and all pesticides) and the introduction of government policies for pesticide reduction, represent an opportunity for the development of new weed control technologies that have reduced risks and are suitable for organic food production (Bailey, 2014).Thus, although only a limited number of isolates were evaluated in the present study, this is the first report that demonstrates pathogenicity of fungal isolates on C. bonariensis in Argentina and represents the first step in a future biocontrol program into integrated weed management. jas.ccsenet.

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Figure 1.O