Cross-protection of cotton against Verticillium wilt by Verticillium nigrescens

doi: 10.9755/ejfa.2015-04-047


INTRODUCTION
Verticillium wilt caused by Verticillium dalhiae Kleb., is a well-known serious disease of many economical crops, especially of cotton where it can reduce cotton yield greatly under certain environmental conditions (Pegg, 1984;Bhat and Subbarao, 1999).The fungus is widespread in most cotton cultivated areas and is one of the greatest threats to cotton production worldwide (Erdogan and Benlioglum, 2010) as well as in cotton areas in Greece where this disease is one of the main constraints to cotton production especially in the region of Thessaly (Tjamos et al., 2000).
Most cotton plants are asymptomatic at initial infection (Shen, 1992;Xiao et al., 1998;Ma, 2007), thus, cotton yield losses can be reduced effectively when cotton Verticillium infection is predicted early and accurately, and prophylactic measures are properly taken (Jing and Huang, 2010).However, according to Paplomatas et al., (1992) when infection occurs at later stages infected plants usually exhibit symptoms of marginal necrosis in leaves, discoloration of the stem vascular bundles, decrease in photosynthesis and increase in respiration, resulting in a significant reduction of the plant biomass and significant loss in yield.
The study of the symptoms on the infected plants depends mainly on the manual investigation and sampling in fields which is time and effort consuming and difficult to practice for large areas.Thus, correlation analysis of leaf spectral reflectance and disease severity of cotton Verticillium has shown that there are a number of sensitive bands that spread in several spectrums (Ning et al., 2013).
In general, different methods of controlling Verticillium wilt of cotton plant like heat treatment, fumigation with broadspectrum biocides and eco-friendly friendly alternative methods like rhizobacteria (Weller, 1998;Fravel and Larkin, 2000;Landa et al., 2004) mycoparasites (Grunden et al., 2001;Rekanovic et al., 2007;Zheng et al., 2011) and endophytes (Yang et al., 2013) have been well-reported.Furthermore, according to Xue et al., (2013) shown that the application of powdered biocontrol agent containing living actinomycetes could be useful for biocontrol of the plant pathogen in agricultural production.The isolates successfully colonized the cotton rhizosphere in a wilt-sick field study when the powdered biocontrol agent was applied either as a seed coating or as a soil inoculation.Moreover, planting resistant varieties is a possible strategy for protection of cotton from V. dahliae, but few upland cotton cultivars showed resistance towards Verticillium wilt (Bolek et al., 2005;Gore et al., 2009;Gao et al., 2011).
Furthermore, organic amendments in applied soil also reduced disease severity in both inoculated pots and naturally infested cotton field plots.According to Huang et al., (2006) the most effective control was achieved with crab shell (chitin), soybean stalk and alfalfa.
The aim of this study was to to determine possible cross protection effects of V. nigrescens against V. dahliae in susceptible cotton cultivars in the region of Thessaly, Greece.

MATERIALS AND METHODS
In the present study the severe pathogen (V.dalhiae) was isolated, in the laboratory of plant protection of Technological Educational Institute of Thessaly (T.E.I.), several times from cotton plants showing symptoms of Verticillium wilt at several locations in central Greece.In order to determine protection against a virulence isolate of V. dalhiae, an isolate of V. nigrescens, a comparatively weaker vascular pathogen, recovered from cotton plants showing weak wilt symptoms, was tested.Following that we tested cross protection effects in cotton against disease induced by a virulent isolate of V. dalhiae with the weaker vascular pathogen V. nigrescens four days prior to inoculation with a challenger strain based on the idea presented (Melouk and Horner, 1975).Moreover, that cross protection effects were tested in process biochemical mechanisms, antifungal mechanisms, such as phytoalexins of cotton plants in response to inoculums of the aggressive pathogen (V.dalhiae) as represented in previous research work (Zaki et al., 1972).
Number of leaf discoloration (foliar damage by 0-5 rating scale where 0: no foliar symptoms; 3: 25% of plants showing evidence of leaf yellow; 5: severe foliar damage), stem vascular staining (darker in color by 1-4 rating scale where 1: light vascular staining to 4: vascular staining darker in color), stem height, diameter, fresh weight and dry weight of inoculated plants were assessed at the end of the experiment, after 45 days of incubation.All data were analysed by analysis of variance (ANOVA) and presented with graphs using Microsoft Excel.
Xylem sap flow investigation in cotton plants infected with or without Verticillium species was determined as follows.
The study was carried out after 40 days of inoculation in eight periods (30 to 240 min, Fig. 4), using two random plants for each period.The probes of phytometric system LPS-03 Phytomonitor (PhyTech Ltd) was established into the stem up to a height of 20 cm from soil surface to measure sap flow density.

RESULTS AND DISCUSSION
The results showed that cotton plants inoculated with only V. nigrescens were symptomless, obtained significant high values for stem height, diameter, fresh and dry weight and significant less values for number of leaf discoloration and stem vascular staining compared to those cotton plants inoculated with only V. dalhiae (Figs.1-3).When inoculation with V. nigrescens precedent V. dalhiae, mild wilt symptoms occurred in all cotton tested plants, agreeing with Korolev and Katan, (1999).Plants which were inoculated on the same day as V. nigrescens before the inoculation with V. dahliae were statistically different from those inoculated on the same day as V. dahliae before the inoculation with V. nigrescens, in number of leaf discoloration, stem vascular staining, diameter, fresh and dry weight (Figs.1-3).
When V. nigrescens was inoculated four days after V. dalhiae, significant wilt symptoms were observed as measurements such as stem diameter, stem height, foliar damage, stem discolouration, stem dry weight and stem fresh weight were done (Figs.1-3).
Moreover, the results of Figs.1-3 also showed that when V. dalhiae was inoculated four days after V. nigrescens, a significant less wilt symptoms was observed, and we can say that V. nigrescens induced a crop protection effect against V. dalhiae when introduced first in cotton plant rhizosphere.
As determined by measurement the transpiration rates of mass flow of sap in the cotton stem (Figs. 4), the rate of mass flow of sap increased significantly in plants inoculated with only V. nigrescens and in plant when V. dalhiae was inoculated four days after V. nigrescens.This is in agreement with previous research work, where cross protection effects were observed in peppermint when inoculation with V. nigrescens preceded inoculation with V. dahliae by two days (Melouk and Horner, 1975).

CONCLUSION
In the present investigation results show that it is possible to reduce disease induced by a virulent isolate of V. dalhiae, when inoculated first with a weak pathogen, V. nigrescens.
It is believed that cross-protection of cotton to a severe isolate of V. dalhiae was possible due to accumulation into xylem of antifungal secondary metabolites in response to inoculation with V. nigrescens, (Zaki et al., 1972;Schnathorst and Mathre, 1996).Finally, we can concluded that the crossprotection phenomenon reported in the present study is a form of a biological control occurred in nature and may have application in control of wilt diseases in plant (Price and Sackston, 1989;Huertas-Gonzalez et al., 1999;Erdogan et al., 2013).

Fig 3 .Fig 4 .a
Fig 3. Cotton stems fresh weight (a) and dry weight (b), in inoculated plants with V. nigrescens and V.dahliae.b