In vitro Methodology to Assess Quantitative Resistance in Plant-fungus Pathosystems

3.1. Infection Efficiency

IE is defined as the probability that a spore deposited on a receptive host surface produces a lesion in the absence of competitive interactions. It is usually measured as a percentage of successful infections resulting from a controlled number of deposited spores [64]. For practical reasons, IE may be indirectly measured by the observed numbers of chlorotic flecks or lesions per unit of leaf area [65]. This trait is difficult to precisely estimate because it depends on the number of spores deposited as well as on microclimatic conditions, which are difficult to control [66].

3.2. Latent Period

LP is the time interval between infection and the onset of sporulation from that infection. It determines the duration of epidemic cycles and thus largely controls the rate of epidemic development. The definition of LP is clear when applied to a single lesion. In most experimental studies, however, artificial inoculations result in many infections per leaf, and the variation in observed LP among infection sites on a leaf may be considerable [67]. To cope with this difficulty, several criteria are used to estimate LP, such as the time from inoculation to first sporulation [68] or the time needed for half of the final number of lesions to sporulate [69] or to show apparent sporulation structures [70]. The most precise method for estimating the time needed for half of the final number of lesions to sporulate was proposed by Shaner [67] and is based on an adjustment of the dynamics of lesion emergence to a sigmoid curve. Since LP is highly dependent on temperature, it is recommended to express the time in degree-days to allow comparisons between different experiments [71]. It has been observed that using different methods to measure LP (i.e., the time needed for half of the final number of lesions to sporulate vs. the time to first sporulation) could lead to differences in its estimation as observed for Puccinia triticina/wheat [65] and Phytophthora infestans/potato [69]. Such differences might, of course, result from uncontrolled environmental effects. A more interesting alternative is that variability in LP among fungal isolates could reveal heterogeneity in both the time at which the first sporulation occurs and the dynamics of lesion maturation [72].

3.3. Sporulation Rate

SR is the number of spores produced per lesion and per unit of time [73, 74]. In practice, spores are either weighed [75] or counted [76]. Sporulation is sometimes expressed in spore production per unit area of diseased leaf [75] or relative to lesion size [76]. Spore production per lesion is highly density-dependent [73]. It can be useful to consider the spore production per unit area of sporulating tissue [76], which is considerably less density-dependent [77]. The number of spores produced by a diseased plant will determine the quantity of inoculum that can infect neighboring plants but also indicates the interaction between the host plant and the parasite since the quantity of spores produced depends on the aggressiveness of the parasite and the level of host QR [4]. It also reflects the capacity of the fungus to invade host tissues during the incubation period.

The infectious period is the time from the beginning to the end of sporulation. This component is difficult to precisely estimate since sporulation often shows an early peak, followed by an asymptotic decrease [77]; however, more irregular patterns may be obtained [78]. For cereal rusts, sporulation can last for more than 40 days under controlled conditions on adult plants [77].

Since many pathogen species have two or more forms of propagule related to sexual or asexual reproduction [79], infection efficiency, sporulation rate, latent period, and infectious period may, in principle, be measured for each type of spore [80]. Nevertheless, a given parameter may not have the same meaning when measured on sexual and asexual spores. For example, the latent period associated with sexual spores is very different from that of asexual spores because it depends on the fortuitous encounter and merger of two sexually compatible lesions. Therefore, while there seems to be room for adaptive adjustment of asexual latency, sexual latency is expected to be highly dependent on environmental stochasticity [1]. Moreover, organs resulting from sexual reproduction often ensure inter-season survival, as in Blumeria graminis or Leptosphaeria maculans, and the latent period, as defined above, has no meaning in such cases [1].

3.4. Lesion Length

LL is another quantitative trait that is measured as a QR component [81]. It is generally defined as the surface area that produces spores. For some pathogens, such as Puccinia triticina/leaf rust, lesion size remains limited, but it can dramatically increase in some species, such as Puccinia striiformis var. striiformis/stripe rust or Phytophthora infestans/late blight, for which lesion growth is semisystemic [82]. In this case, LL accounts for a large part of the quantitative development of epidemics and lesion growth rate is a key factor in pathogen competition for available host tissue. LL is not easy to precisely determine for pathogens, such as Mycosphaerella graminicola/Septoria blotch, that induce necrosis on the host leaf [83]. Moreover, such pathogens often indirectly cause apical necrosis on the leaves that can be confused with a diseased area.

3.5. Dwarfing

Dwarfing is a symptom characteristic of plants systemically infected by fungal pathogens and is explained by a decrease in the concentration of growth hormone in infected tissue [4]. This decrease in size can be observed at a very early stage.

3.6. Disease Severity

QR is sometimes estimated through disease severity, measured as the percentage of the infected plant organ (root, leaf or fruit/spike) covered by pathogen lesions [84]. DS is a composite variable resulting from the integrated effect of infection efficiency and lesion size, but also, when assessed at the crop scale, sporulation and dispersal.

In any case, it is essential to measure quantitative traits that can be clearly related to disease dynamics and pathogen evolution. For instance, a few in vitro traits have been identified that may be useful for the characterization of quantitative resistance by making comparisons with two cultivars that are susceptible to the pathogen in the field. The collective effects of each or some of the in vitro traits are particularly important.

4.1. A Necrotrophic Fungus

A detached leaf assay was used to determine the reaction of five barley genotypes to spot blotch caused by Cochliobolus sativus [25]. The estimation of the infected leaf area enabled discrimination among barley genotypes differing in their susceptibility to the pathogen. Significant correlations were found (P=0.001) between in vitro values in both seedling (r=0.89) and adult (r=0.95) plants. The established in vitro assay enabled a fast assessment of the susceptibility of barley to spot blotch and should be useful for many types of studies on this disease [25].

4.2. A Hemibiotrophic Fungus

The detached leaf assay was successful in the identification of an important component of the resistance of Fusarium species, causing head blight (FHB) in the European wheat germplasm. This in vitro method may be a useful mechanism to discriminate amongst different types of resistance in a breeding program [8]. Michodochium majus is used in the detached leaf assays to detect leaf symptoms for observing QR components (incubation period, latent period and lesion length) [12]. Lesion length is one of the components of QR measured as an indicator of fungal pathogenicity and aggressiveness. QR components detected in the M. majus detached leaf assay have been correlated to FHB resistance in wheat inoculated with F. culmorum and F. graminearum [8, 12]. FHB resistance in a seed germination assay was highly correlated in F. graminearum, F. avenaceaum, F. culmorum, M. majus and Microdochium nivale, indicating common resistance between M. Majus and other Fusarium spp. in the in vitro assay [12]. Browne and Cooke [13] reported that QR components (incubation periods, longer latent periods and shorter lesion lengths) in the detached leaf assay and higher germination rates in the seed germination assay were related to greater FHB resistance (Type II). However, the exotic wheat germplasms, which provide highly effective resistances to FHB resistance, do not appear to be detected in the detached leaf or seed germination assays [8, 12].

4.3. A Biotrophic Fungus

In vitro components of quantitative resistance in sunflower (Helianthus annuus) to Plasmopara halstedii [47], i.e., percentage infection, latent period, sporulation density, and reduction of hypocotyl length, were compared between two sunflower cultivars and showed different levels of quantitative resistance in the field. The moderately susceptible inbred line showed a higher percentage of infection, a higher sporulation density, a shorter latent period and less reduced hypocotyl length than the moderately resistant inbred line. The percentage infection of FU was 1.4% less than BT, the latent period of BT was 12.4% less than FU, the sporulation density of FU was 22.3% less than BT, and the reduced hypocotyl length of BT was 15.3% less than FU. It seems that the criteria, such as latent period, sporulation density and reduction of hypocotyl length, may be used to measure quantitative resistance in sunflowers to P. halstedii [47].

The above-mentioned studies have generally concluded that in vitro traits can be applied for measuring QR in several plant species infected with biotrophic, hemibiotrophic, and necrotrophic fungal pathogens.

5.1. Growth Stage

The level of expression of QR is known to vary with plant development [3, 4]. For example, QR is greater in late expanding leaves against ascochyta blight in chickpeas and faba beans [33, 37], powdery mildew and crown rust in oat [85, 86], powdery mildew in sweet cherries and rusts in grapes [40, 41], rust and Fusarium head blight wheat [87, 88], powdery mildew in leaf blight in taro [56] and southern corn leaf blight in maize [28]. The effect of leaf position was considerably more pronounced in the powdery mildew-grapevine interactions; the magnitude of differences between susceptible and resistant cultivars increased with leaves at positions 3-5. Data suggested that leaves at position 3 must be inoculated to clearly distinguish resistant from susceptible cultivars [41]. Senescing grapevine leaves became more resistant to grapevine powdery mildew, and this effect was evident in both the success of infection and the diameter of the colony [41]. Conversely, older cucumber leaves were more susceptible to gummy stem blight than younger leaves in the field, greenhouse, and detached-leaf tests [7]. Arraiano et al. [18], using a detached leaf assay in wheat, found that secondary leaves were more susceptible to Septoria tritici than first-expanding leaves. Important factors for downy mildew/Plasmopara viticola resistance screens include leaf age; the fourth fully expanded leaf corresponds well with vineyard ratings [89].

5.2. Senescence of Plant Parts

A critical aspect of the in vitro assay is the prevention of senescence of leaf pieces before the duration required to express symptom stages and disease severity levels necessary to differentiate among cultivar responses. The leaf senescence may have had an inhibiting effect on sporulation for a period before sporulation occurred profusely over leaf stomata [41]. The youngest tissues can be infected, but the development of the fungus is generally arrested during the aging of tissues before sporulation occurs [41]. Senescence of leaf tissue before symptom development altered the accuracy with which QR resistance was determined, which has implications for using detached leaf assays to determine resistance to other diseases [88]. Using whole plants, which prevents the senescence associated with detached leaves, Singh and Huerta-Espino [87] found that cultivar differences were most clearly observed in later-forming leaves, which showed greater resistance. In a detached leaf assay, Arraiano et al. [18] inoculated seedling leaves with an S. tritici, which showed a relatively long period between inoculation and symptom development prior to detaching the leaves and mounting them on water agar.

5.3. Amending Growth Medium With Plant Hormones

Amending agar medium with plant hormones aided the retention of green leaf color in detached leaves [9, 12, 26, 90]. Benzimidazole and cytokinin have been used in the incubation medium to prevent chlorophyll degradation in detached leaves. Detached wheat and barley leaves were kept green for 14 to 34 days on benzimidazole agar [26], detached wheat leaves were kept green for at least 14 days using kinetin [12], and detached oat leaves were kept green for up to 10 days using kinetin and 6-benzylaminopurine treatments [86], indicating that maintaining green detached leaves for much longer periods is essential to propagate fungi. Soybean leaf pieces in a medium containing kinetin at 10 mg/liter had 5% chlorosis 18 days after plating compared to leaf pieces in media amended with all other plant hormones, which had higher levels of chlorosis [9]. Longer periods of healthy leaf tissue are important for the short-term use of rust isolates [90].

5.4. Incubation Conditions

The incubation conditions of detached potato leaves in closed trays rather than detachment itself appeared to affect the late blight resistance expression, concluding that the lower expression of resistance in the detached leaf tests was due to differences in environmental conditions [45]. The constant, highly favorable environment a pathogen finds in the closed trays apparently enhances infection by the Phytophthora infestans zoospores. Incubating detached leaves in closed trays appears to decrease resistance expression. Therefore, a suitable experimental condition must be chosen depending on the aim of the experiment. When the expression of resistance is to be examined on detached leaves, the reduced level of resistance should be measured against the low infection frequency inherent to intact plants. Inoculation of intact plants is preferred, but in most cases, the inoculation of detached leaves incubated in covered trays appears to be an adequate alternative [45]. Relative humidity during incubation in closed containers was 85% and may have proved lethal to the more fragile Phytophthora colocasiae zoospores (taro leaf blight) without an agar drop to protect them [56].

5.5. Origin Of Genetic Host And Fungal Materials

The origin of genetic host and fungal materials plays a critical role in evaluating in vitro resistance reactions. Brown and Cooke [12] did not find a relationship between the detached leaf assay and whole plant FHB resistance in International Maize and Wheat Improvement Center (CIMMYT) genotypes containing more diverse sources of resistant germplasms in their pedigree. The detached leaf assay did not appear to detect the whole plant resistance of Sumai 3, a Chinese wheat cultivar, because it displayed shorter incubation and latent periods and longer lesion lengths relative to European cultivars. Moreover, the detached leaf technique did not detect the greater FHB resistance of Frontana wheat cultivar because this genotype had a comparable incubation period, latent period, and lesion length to the most resistant Irish and UK cultivars possessed only moderate FHB resistance [12]. Although considerable differences in mean Plasmopara viticola/downy mildew severity separated Vitis vinifera, Vitis hybrid, V. riparia, and V. labrusca during in vitro inoculations (from susceptible to resistant), notable intra-species variation was identified for all well-represented species [89]. The effect of the pathogen source on resistance ratings could reflect genetic variation in P. viticola; the lack of disease severity was much more likely due to resistance against the single isolates screened [89].

Certain precautions are required while conducting detached-leaf assays. Leaves should be free from mites because they forage on rust spores, which in turn, reduce the efficiency of infection. Surface sterilization of leaves with 0.1% NaOCl or 70% ethanol should also be avoided because it causes patches of necrosis that interfere with pathogen development [9].

7.1. Studying Plant-Fungal Interactions At The Physiological Level

Production of single-strain cultures of obligate pathogens for use in genetic studies is difficult. By using in vitro techniques, the ability to handle and propagate pathogens on the young plant materials helped to generate pure cultures and avoid cross-contamination for Podosphaera fusca, causing cucumber powdery mildew, Pseudoperonospora cubensis causing cucumber downy mildew, Plasmopara halstedii causing sunflower downy mildew, Puccinia coronata causing crown rust, Phakopsora pachyrhizi causing soybean rust, and Peronospora effuse causing spinach downy mildew [48, 86, 90, 97, 98]. As a pre-screening test, the effect of potential resistance-inducing chemicals, DL-3-aminobutyric acid, Bion (benzo-(1,2,3) thiadiazole-7-carbothioic acid S-methyl ester), and a foliar fertilizer containing potassium phosphite, on development of Microdochium majus causing Fusarium head blight was studied in detached leaves [99]. Reduced disease development of M. majus was also only observed in detached leaves pre-treated with the foliar fertilizer, indicating that the effect on disease development was at least partly due to a fungistatic effect [99]. The in vitro technique also performed well in testing the pathogenic behavior of Erysiphe necator on grapevine [41], Plasmopara halstedii on sunflower [100, 101], Mycosphaerella fijiensis on bananas [30], Phytophthora infestans on potato [102], Fusarium and Microdochium species causing a head blight on wheat and barley [16, 17, 103-105] and Phytophthora cactorum, P. citrophthora, Pythium dissotocum complex, Py. aphanidermatum, Globisporangium heterothallicum, G. ultimum, Phytopythium vexans, Phy. mercurial, and Phy. litorale on strawberries [106]. The in vitro screening method was effective in determining the pathotype of Pseudocercospora griseola isolates on a set of angular leaf spot differential common bean cultivars [107] and host-specialization in E. necator-V. vinifera interactions [41] and four Fusarium species causing a head blight on wheat and barley [108-110]. The in vitro laboratory assay was used efficiently to determine the sensitivity of P. infestans and two rust fungi to fungicides [111, 112]. Phytotoxins as selective agents under in vitro selection were utilized for improved disease resistance [113].

7.2. Studying Plant-Fungal Interactions At The Molecular Level

The detached leaf assay provided a means to enable fundamental studies on the defense mechanisms of Arabidopsis in response to F. graminearum/Fusarium head blight [114]. In vitro infection of detached leaves Puccinia substriata, the causal agent of rust disease, and Sclerospora graminicola, the causal agent of downy mildew, resulted in a significant reduction of disease symptoms in transgenic pearl millet cultivars in comparison to wild-type control plants; the disease resistance of pearl millet was increased by up to 90% when infected with two diverse, economically important pathogens [115]. On detached leaves, virus-induced gene silencing was adopted to dissect gene functions in cotton resistant to Verticillium wilt [116]. Using in vitro methods, transgenic lines of tomato harboring rice chitinase gene were evaluated for resistance to Fusarium oxysporum f. sp. lycopersici causing Fusarium wilt and Alternaria solani causing early blight. In vitro studies concluded that introducing a chitinase gene in a susceptible cultivar of tomato not only enhanced the resistance but was stably inherited in transgenic lines [117]. Using detached leaf assay, two novel powdery mildew resistance loci, Ren6 and Ren7, were identified from the wild Chinese grape species Vitis piasezkii [118]. Phenotyping of transformed potato cultivars developed through small interfering RNA (siRNA) and artificial micro RNA (amiRNA) techniques for late blight resistance was undertaken using in vitro assays. The siRNA and amiRNA-derived cultivars exhibited less lesion area as well as spore production and were categorized as moderately resistant [41].

7.3. Using In Vitro Techniques To Explore Defense Responses In Plants To Fungal Infection

Wheat endophytes, Phoma glomerata, Aureobasidium proteae and Sarocladium kiliense, were screened as biological control agents against Fusarium head blight using two different in vitro tests [119]. This study pointed out that the test on detached wheat spikelets provided information about the potential pathogenicity, growth capacity, and efficacy of the endophyte strains on the targeted plant before testing them on whole plants [119]. Detached leaves of the Arabidopsis lines were inoculated with Verticillium dahliae to evaluate the defense response under drought stress; leaves from transgenic plants showed increased callose deposition and reduced mycelia growth [120]. The effect of chemical treatments affecting the ethylene pathway was studied in detached head assays, and the results showed that the ethylene signaling could mediate wheat Fusarium head blight resistance to Fusarium graminearum [121]. A notable difference was found between non-amended and amended treatments with silicon on young plant parts in potato detached leaves infected with Phytophthora infestans (late blight) [122] and wheat detached leaves and seedlings challenged with four Fusarium head blight pathogens [123]. By in vitro techniques, the direct fungitoxicity was investigated for Fusarium head blight pathogens and Cochliobolus sativus causing spot blotch and common root rot [124]. Recently, Sakr [125, 126] explored how the deployment of QR in wheat and barley affects aggressiveness under in vitro conditions, leading to potential resistance erosion.

7.4. Using In Vitro Techniques In Plant Breeding

In breeding programs and during gene pyramiding, it will be challenging to evaluate plant symptoms using only conventional screening methods; however, this alternative rapid detached leave method is proposed for marker-assisted gene pyramiding in combination with molecular markers to aid the selection of resistant progenies during each backcross generation [107]. In addition, the proposed method can help when evaluating progenies for different pathotypes using leaves from the same plant, which could be difficult with the conventional evaluation method. Thus, this alternative screening technique could provide a great opportunity for breeders working in gene pyramiding and marker-assisted back-crossing programs to evaluate common bean genotypes and progenies for multiple disease resistance [107].