Detached vs. attached leaf assays: summary of discussion
In 2008, Alice Churchill initiated a discussion on conducting plant bioassays with banana pathogens. Her lab had recently described a quantitative assay using mycelial fragments to assess virulence of Mycosphaerella fijiensis on intact plants under controlled growth chamber conditions; they had not had success using a detached leaf assay (Donzelli and Churchill, 2007). She referred to an article by Liu et al. (2007) that reported significant differences in plant defence responses when Colletotrichum was assayed on detached vs. attached leaves of Arabidopsis. Symptom development in detached leaves appeared uncoupled from typical plant defence response pathways and more closely associated with responses involved in plant senescence, demonstrating that the host-pathogen interaction in detached leaves was atypical. Alice raised the question of whether banana bioassays to evaluate disease development by M. fijiensis using detached leaves (pieces) rather than attached leaves on intact plants produce disease symptoms and plant responses comparable to those observed using intact plants. She argued that the validation and standardization of greenhouse, growth chamber or lab bioassays that will reliably predict plant disease responses in the field is critical for accurately evaluating banana resistance and susceptibility, as well as virulence of diverse pathogen populations.
Andrew James responded that he has had doubts about detached leaf assays since his experience with Hedera helix in which accumulation of ethylene was one of the factors giving artifactual results at the molecular level. While his team is not abandoning the detached leaf assay, he agrees that it needs to be evaluated more. His team also uses intact plants for transient iRNA assays. Andrew suggested that evaluating the detached leaf assay at the molecular level should be a priority for the international banana research community.
Yelenis Alvarado-Capó referred to Townley et al. (2001) who used visual observations and scanning electron microscopy to show that the development of disease is significantly different in detached and intact leaves. Twizeyimana et al. (2007), on the other hand, conclude that two assays using in-vitro plantlets in tubes and detached leaves are rapid and space effective, and can reliably be used for screening Musa genotypes for resistance to black leaf streak. Alvarado-Capó et al. (2003) standardized a protocol for early screening of Musa genotypes in the greenhouse using mycelial fragments of M. fijiensis cultivated in liquid medium. The method has been used for plant-pathogen interaction studies. They also tested the detached leaf and in-tube assays, but the results could not be compared with the greenhouse assay. The critical aspects for the development of the disease, like inoculum density, genotype reaction, isolate influence, as well as components of resistance in the greenhouse were studied too. The team was willing to offer their experience and cooperation if anyone is interested in other studies.
Luis Perez-Vicente responded that one needs to use mature expanded leaves to get a realistic response. Symptom expression is different in leaves of young tissue-culture plants than in expanded leaves of plants in the field, and in most of the cases the in-vitro plantlets are more susceptible. He further argues that we have to focus on what we want to demonstrate. For banana breeding purposes, these kinds of tests can be used for early screening to discard very susceptible genotypes.
Luis further talked about complications when conducting aggressiveness/virulence studies with different isolates of M. fijiensis, especially when quantitative responses are involved and we want to avoid the influence of the physical environment on these responses. Luis and his team have used the detached leaf assay to compare aggressiveness of two M. fijiensis populations following some epidemiological principles, such as: 1) spore load by unit of leaf surface should be in a range where there is a linear increase of infection as well as independence in the infection (not positive or negative interaction) between spores; 2) spore load should agree with natural spore loads observed on leaf surfaces in the field according to published research; 3) adjusted curve of number of lesions per number of spores/cm2 should go through the origin; 4) there should be agreement between the curves in the leaf fragment assay using a set of standard cultivars of known reaction and the order of response in the field with those cultivars; 5) there should be correspondence between the incubation and transition periods between the detached leaf fragment assay and the field assay for the different cultivars of different known reaction. With these premises, we can compare the aggressiveness of different isolates and establish differences between them.
Mauricio Guzmán agreed with Luis’ comments. Another important limitation he sees with the detached leaf assay is the break-up of the relationship between the root system (mineral and organic nutrition, water uptake, soil microorganisms, etc) and the foliar system (photosynthesis, transpiration, foliar emission, etc). The importance of this relationship in the host-pathogen interaction, not only for M. fijiensis, has been shown several times.
Elio Jiménez González and his team decided to use the intact plant method, developed by Alvarado et al. (2003), for molecular studies on M. fijiensis-Musa interactions, based on the idea that detached leaves could display an array of responses not solely related to the reaction against pathogen infection but also to general stress and senescence. They constructed two subtracted libraries from compatible (Grande Naine) and incompatible (Calcutta 4) interactions and a full length cDNA library from the compatible interaction (Grande Naine). No sequences related to leaf senescence were identified so far, although the number of sequenced clones is still limited. Yelenys also described the application of the intact plant assay to screen transgenic plants expressing antifungal proteins, corroborating the results of field trials (publication in preparation). On the other hand, as Luis mentioned, the detached leaf assay could be useful for other studies, i.e. to check the aggressiveness of different isolates.
Ranajit Bandyopadhyay agreed that the choice of evaluation method depends on the objectives of the study, with all methods having their own advantages and disadvantages. Lieberei (2007), studying South American leaf blight on rubber, found that scopoletin-related defence reactions are dissimilar in detached vs. attached leaves and the author suggested that attached leaf evaluation should be used for resistance evaluation. However, Chen et al. (2006) found the detached leaf assay to be useful to study defence response genes associated with jasmonic acid/ethylene and salicylic acid pathways in Arabidopsis-Fusarium (a necrotroph) interactions (similar pathway as in the Liu study). Twizeyimana et al. (2007) used banana genotypes with known field reaction, and found that resistance levels in detached leaf and field reaction were similar. They recently concluded studies on Mycosphaerella variability and classical inheritance coupled with molecular basis of resistance (phenotyping segregating materials in detached leaves and host genotyping of attached leaves). Due to the large-scale nature of evaluations, they opted for the detached leaf method for resistance evaluations. In the pathogen variability studies, there were no significant differences in disease reactions between the two runs, suggesting that the test was repeatable. At IITA, a combination of detached and attached leaf assays are used depending upon the objectives of various studies. In the breeding program, the initial disease evaluations are done in detached leaves from potted plants, and segregants with moderate to high levels of resistance are then transplanted to the field to select for desirable agronomic characters and verify disease resistance.
Ranajit further said that banana trials, in field or greenhouse, require a lot of space and time. When a large number of plants are to be evaluated within a short span of time in a limited space under uniform environmental conditions without interference from other pathogen populations, the detached leaf technique can be useful. As Twizeyimana et al. (2007) demonstrated, the detached leaf technique can be used initially as a proxy, but the responses must be verified with the attached leaf assay whenever possible. Unless greenhouses have an excellent weather control system, growing plants in the greenhouse under conditions similar to field conditions is not easy. Nevertheless, the attached leaf greenhouse evaluation proposed by Alice’s group (Donzelli and Churchill, 2007) is very useful since symptom development is similar to what is observed under field conditions. Also, as weather conditions vary from location to location and from time to time within a location, disease reactions in the field may sometimes be difficult to interpret.
Ranajit agreed that it is necessary to have uniform protocols for disease evaluations so that the results are comparable across studies conducted at various locations. He therefore welcomed the idea of comparing various resistance evaluation methods in several labs.
Lara Olaniyi noted that plant phenology (growth stage) has a great influence on susceptibility. She argues that when plant genotypes are screened, their response (susceptible, tolerant or resistant) should be related to yield. She suggests that for pathogenicity assessments, we make comparative studies initially to identify which phenological stage best correlates to yield. Subsequently, screening could then be done at such identified stage to ensure that the assessment relates to the expected final product.
Alvarado-Capó, Y., M. Leiva-Mora, M.A. Dita-Rodríquez, M. Acosta, M. Cruz, N. Portal, R. Gómez-Kosky, L. García, I. Bermúdez and J. Padrón. 2003. Early evaluation of black leaf streak resistance by using mycelial suspensions of Mycosphaerella fijiensis. In Mycosphaerella Leaf Spot Diseases of Bananas: Present Status and Outlook. Proceedings of the 2nd International Workshop on Mycosphaerella Leaf Spot Diseases, San José, Costa Rica, 20-23 May 2002 (L. Jacome, P. Lepoivre, D. Marin, R. Ortiz, R. Romero and J.V. Escalant, eds). Montpellier, France: INIBAP, pp. 169-175.
Chen, X., A. Steed, C. Harden, and P. Nicholson. 2006. Characterization of Arabidopsis thaliana-Fusarium graminearum interactions and identification of variation in resistance among ecotypes. Molecular Plant Pathology 7: 391-403.
Donzelli, B.G.G. and A.C.L. Churchill (2007) A quantitative assay using mycelial fragments to assess virulence of Mycosphaerella fijiensis. Phytopathology 97: 916-929.
Lieberei, R. 2007. South American leaf blight of the rubber tree (Hevea spp.): New steps in plant domestication using physiological features and molecular markers. Annals of Botany 100: 1125-1142.
Liu, G., R. Kennedy, D.L. Greenshields, G, Peng, L. Forseille, G. Selvaraj, and Y. Wei. 2007. Detached and attached Arabidopsis leaf assays reveal distinctive defense responses against hemibiotrophic Colletotrichum spp. Molecular Plant-Microbe Interactions 20:1308-1319.
Townley, A., Foundling, J., Corsten, M., and Pain, N. A. 2001. Mycosphaerella fijiensis disease development in leaves on whole plants and in a detached leaf assay. In: Caribbean Division Meeting of the American Phytopathological Society. Publication no. P-2002-0119-CRA.
Twizeyimana, M., Ojiambo, P. S., Tenkouano, A., Ikotun, T., and Bandyopadhyay, R. 2007. Rapid screening of Musa species for resistance to black leaf streak using in vitro plantlets in tubes and detached leaves. Plant Dis. 91:308-314.