A Quantitative Assay Using Mycelial Fragments to Assess Virulence of Mycosphaerella fijiensis

Abstract

We describe a method to evaluate the virulence of Mycosphaerella fijiensis, the causal agent of black leaf streak disease (BLSD) of banana and plantain. The method is based on the delivery of weighed slurries of fragmented mycelia by camel's hair brush to 5-by-5-cm areas on the abaxial surface of banana leaf blades. Reliable BLSD development was attained in an environmental growth chamber with stringent lighting and humidity controls. By localizing inoculum onto small areas of large leaves, we achieved a dramatic increase in the number of strains that can be tested on each leaf and plant, which is critical for comparing the virulence of numerous strains concurrently. Image analysis software was used to measure the percentage of each inoculated leaf section showing BLSD symptoms over time. We demonstrated that the level of disease of four isolates was correlated with the weight of the mycelium applied and relatively insensitive to the degree of fragmentation of hyphae. This is the first report demonstrating that weighed mycelial inoculum, combined with image analysis software to measure disease severity, can be used to quantitatively assess the virulence of M. fijiensis under rigorously controlled environmental conditions.

Expression of a rice chitinase gene in transgenic banana (‘Gros Michel’, AAA genome group) confers resistance to black leaf streak disease

“…Twizeyimana et al (2007a) concluded that a GA 3 concentration of 5 mg L −1 is adequate to significantly reduce the chlorosis on banana leaf disks, but they observed only 52 days of incubation. Similar to a greenhouse test where successful disease development can take longer incubation period [2–3 months (Churchill 2010; Donzelli and Churchill 2007)], we have found that an 8 mg L −1 GA 3 supplement provides sufficient longevity for leaf disks to fulfill this condition without deteriorating. Although there are suggestions in other plant/fungus interactions (Takahashi et al 2005; Yamamoto et al 2000) that transgene expression just postpones symptom development and prolonging the incubation time results in similarly severe symptoms in transgenic plants as in non-transgenic controls, we observed this phenomenon only in the most susceptible transgenic line (GM.RCG3.09) during our LDA (up to 108 dpi).…”

“…During the last decade, these bioassays became a useful tool for screening transgenic plants for disease resistance (Kumar et al 2003; Marchant et al 1998; Takatsu et al 1999). Two main categories of LDA can be distinguished, detached (Abadie et al 2009; Agnola et al 2003; Bussey and Stevenson 1991; Cohen 1993) and attached LDAs (Donzelli and Churchill 2007; Meeley et al 1992; Twizeyimana et al 2007a, b). In case of banana, rapid mass screening with attached leaves is difficult to carry out because of requirements in space and screenhouse conditions (e.g., high humidity and temperature) for efficient control of inoculations.…”

Analysis of the leaf transcriptome of Musa acuminata during interaction with Mycosphaerella musicola: gene assembly, annotation and marker development

“…[40,41]), with development of disease symptoms not always correlating with those typically observed on intact plants. Additionally, [40] suggested that the hemibiotrophic Mycosphaerella banana pathogens require healthy banana plants for disease development, with [42] suggesting that the physiological state of detached leaves is not comparable with those on whole plants with an intact root system.…”

Singlet Molecular Oxygen Generation by Light-Activated DHN-Melanin of the Fungal Pathogen Mycosphaerella fijiensis in Black Sigatoka Disease of Bananas

“…As we show in figure 5D, the mycelium of the dark strain ( Mf-1 , Figure S3) generates more 1 O 2 than the albino strain ( 102 , Figure S3); therefore 1 O 2 generation by melanin becomes a condition that causes oxidative stress and host cell death. Albino strains of M. fijiensis , compensate for melanin deficiency producing carotenes and higher catalase activitiy (unpublished results) and can infect banana leaves, but the damage is not so severe [21]. It has been long observed that banana plants infected with black Sigatoka are less severely affected in the shade than in full sunlight, suggesting the production of light activated toxins by these fungi.…”