Studies on the pathogen, epidemiology and control of gentian brown leaf spot caused by Mycochaetophora gentianae

Brown leaf spot disease causes significant economic losses on gentian. This report summarizes results from studies conducted in a total of seven years (2000-2003 and 2007-2009) at Iwate Agricultural Research Center in Kitakami, Iwate Prefecture, with special reference to the pathogen, epidemiology and control of the disease. I Symptoms and epidemics of the disease 1. Early leaf symptoms on gentian cv. Jobanni and cv. Ihatovo include minute, grayish white lesions on the upper surface. The lesions progressively enlarge to ca. 5 mm in diameter with yellowish marginal area, and subsequently become brown and rotten. Cv. Ihatovo developes the lesions on both sides of the leaf. Symptoms on cv. Aoi-kaze and cv. Koromokawa-princess, which are brown, circular and conspicuous, are different from the typical symptoms (grayish white, powdery lesions) on cv. Jobanni. 2. Field observations revealed epidemiologic characteristics of the disease as follows. Lesions first appeared in early August, gradually increased toward mid-August, and rapidly between late-August to early-September. The disease was more serious on plant leaves facing between two rows, and on the lower leaves. In the field, lesions were at first found sporadically, and then increased to neighboring healthy plants. II Taxonomic position of the causal fungus 1. The causal fungus (K18) isolated from Gentiana triflora was identified as Mycochaetophora gentianae because of its identity to the the ex-type strain (MAFF 239231) in terms of cultural characteristics, pathogenicity, conidial morphology, and genetic similarity as follows. (1) Cultural characteristics: the isolate grew at temperatures from 5 to 30 degC, but did not grow above 32.5 degC with an optimum at ca. 20 degC. (2) Pathogenicity: inoculation tests showed that the isolate produced lesions on G. triflora cv. Ashiro-no-aki, but not G. scabra cv. Ashiro-no-sawakaze. (3) Morphological characteristics: the isolate produced besom-like sporophores on diseased leaves. Conidial morphologies were almost identical between the two isolates except for their shape of the apex. (4) Genetic similarity: ITS sequence was identified between the two isolates with 99.6% similarity (two nucleotides difference). 2. Microscopic observations revealed that conidia of M. gentianae were produced blasticaly from short conidiophores and detached in a schizolytic manner, leaving unthickened and inconspicuous scars on the conidiogenous cells. 3. Phylogenetic analyses revealed the phylogenetic position of M. gentianae and its allied fungi, Pseudocercosporella-like hyphomycetes in conidiogenesis and conidial morphology. (1) Phylogenetic analyses using 3 rDNA sequences combined (SSU + LSU + 5.8S rDNA) indicated that M. gentianae was a member of the Helotiales-Rhytismatales clade with strong support (BP = 88%) and placed in proximity to the helotialean families, but failed to characterize the position of M. gentianae at the family level. (2) Molecular and morphological data clearly showed that M. gentianae had affinity with three helotialean, Pseudocercosporella-like hyphomycetes (Helgardia, Rhexocercosporidium, Rhynchosporium) but that the fungus was distinct from these fungi. (3) ITS phylogeny indicated that M. gentianae was also related to Cadophora, Leptodontidium, and Pyrenopeziza brassicae, as well as helotialean Pseudocercosporella-Iike hyphomycetes. III Physiological characteristics of the causal fungus 1. Conidium germination (1) Conidia germinated at temperatures between 10-30 degC with an optimal range between 20-25 degC and at pH 3-9 with an optimal range between pH4-9. There was no difference in the percentage of conidium germination between on WA and potato dextrose agar (PDA). (2) Relative humidity (RH) over 99% was required for conidium germination (the optimum was 100% RH). The rate of conidium germination was higher in dry-water than in free-water treatment at 100% RH. 2. Mycelial growth (1) Colony growth of the fungus was vigorous on PDA, potato carrot agar (PCA), V8-juice agar, oat meal agar, malt extract agar, and yeast extract agar. Colonies grown on both V8-juice agar and oat meal agar were yellow to olive. Felty colonies developed on yeast extract agar. The fungus failed to produce conidia on these tested media. (2) Colony grew on PDA adjusted at pH 3-10 with an optimal range between pH4-10. 3. Induction of conidium formation (1) PCA slide culture induced conidium (sporophore) formation. Sporophores were formed on the mycelia grown over the surface of cover-glass and on extended growth beyond cover-glass. Additionally, sporophore formation was induced by removing cover-glass or cellophane overlaid on PCA-slide culture. (2) Conidial suspension > 10E5 conidia/ml was obtained when potato-carrot broth inoculated with mycelial blocks was shaken at 120 rpm at 15 degC for 5 d. IV Epidemiology 1. Effect of inoculum density, temperature, leaf wetness duration, and leaf age on infection of the causal fungus with conidia Inoculation tests with conidial suspension revealed that disease incidence was affected by inoculum density, temperature, and leaf wetness duration. (1) Inoculum density: disease incidence increased with increasing inoculum density. Even an inoculum density as low as 10E2 conidia/ml could produce lesions. Logarithm of inoculum density was closely correlated to disease incidence (R**2 = 0.840). (2) Temperature: temperatures between 15-25 degC were conducive to the disease, but those below below 10 degC did not cause any lesions. (3) Leaf wetness duration: disease incidence increased with the increasing duration of leaf wetness (36-72 h), but no lesions occur when leaf wetness lasted less than 24 h. Leaf wetness duration required for developing more than 50% of disease incidence was 60, 48, and 36 h at 15, 20, and 25 degC, respectively. (4) Leaf age: disease incidence on the top (youngest) leaves was lower than on the middle and bottom leaves (oldest), although analysis of variance indicated that the F value of leaf age was lower than that of temperature and leaf wetness duration. 2. Invasion of gentian leaves (1) On gentian leaves, conidia germinated well in the range from 20 deg to 25 degC. After 48 h of incubation, all conidia germinated at 15 deg-25 degC. (2) Lesions that developed 7 d after inoculation had one to several germinated conidia. The conidia generally had germ tubes from one or both ends of the cells, but some germinated from the middle of conidial cells. Appressoria were usually formed at the tip of germ tubes and varied in shape, color and size, i.e., they were often clavate to ellipsoid, hyaline to pale brown and 8-10 * 5-8 micro m. It is notable that appressoria were formed near the grooves on the boundary of epidermal cells. Hyphal mat developed under epidermal cells of the lesions. (3) Appressorium formation was markedly influenced by temperature and leaf wetness duration. More appressoria were formed at higher temperatures (15-25 degC) with extended duration of leaf wetness (24-72 h). At 48-h leaf wetness, the rate of appressorium formation was 0, 8, 26, and 73% at 10, 15, 20, and 25 degC, respectively. 3. Latent period for disease development (1) When inoculated plants were maintained in a moist chamber at 25 degC, minute lesions appeared 7 d after inoculation, and then developed conspicuously 14 d after inoculation. (2) The latent period for disease development was longer at lower temperature (15-20 degC) as compared with 25 degC. 4. Conidium formation and dispersion on diseased leaves Sporophores were produced on diseased leaves when they were incubated in a moist chamber for several days. The sporophore formation was affected by (1) disease severity of the leaf tissue, (2) growth stage of host plants, (3) temperature, and (4) relative humidity (RH). Conidia produced on diseased leaves were easily dispersed into water drop. (1) Disease severity of the leaf tissue: sporophores were formed exclusively on brown and rotten lesions. (2) Growth stage of host plants: the rate of sporophore formation on diseased leaves was higher in the early stage (May-June) and in the blooming stage (September-October) than in the budding stage (July-August). (3) Relative humidity (RH): over 99% RH (the optimum 100% RH) was required for sporophore formation; RH below 86.5%, however, prevented even conidiophore formation. (4) Conidial dispersion: conidia were dispersed from the sporophores into water drop. The number of conidia in the droplet was the highest at 20 degC (6.2*10E2 conidia/ml) when diseased leaves were incubated in the humidity for 3 days; it was 4.2 * 10E4 conidia/ml when incubated at 25 degC for 4 days. 5. Primary inoculum source (1) Neither conidiophore nor sporophore was found on overwintered, infected leaves in an unglazed pot. Conidiophores emerged from the overwinetered, infected leaves when they were incubated into a moist chamber at 15 degC, and subsequently sporophores developed with numerous conidia. Sporophores were formed on the overwintered leaves sampled by July; the frequency of the sporophore formation was the highest on the leaves sampled in April, then became lower thereafter with increasing air temperature. Sprophores were not found in August. (2) Plants were diseased when planted in spring in the soil infested the previous year or when debris of diseased plants had been laid on the soil surface the previous fall. Even though the diseased debris was removed before planting gentians, lesions developed on the plants. The removal of the debris decreased the number of lesions. The lesions were more abundant on the lower leaves than upper leaves. (3) Cultures were obtained from lesions on inoculated plants using the debris of the overwintered diseased leaves and used for PCR detecting with SSU rDNA. The size of PCR products agreed with that of the original culture (isolate K18) with a length of ca. 1600 bp but differed from that of the reference culture (isolate J4) with ca. 1000 bp. (4) When healthy gentians were planted in soil contaminated with conidial suspension, the disease never occurred in 2008, but only a slight number of lesions were found in 2009. Further studies are required for elucidating the soil-born nature of the pathogen. 6. Infection process in commercial fields (1) Four-year exposure tests (2001-2003 in Hanamaki and 2008 in Kitakami) revealed that the infection of the causal fungus started in late June to early July and continued until September in the commercial fields. Inoculum potential was highest in early July before disease occurrence, and after disease occurrence, it culminated in mid to late August. Disease incidence of plants exposed in both periods tended to be enhanced by many rainy days. (2) Multiple regression analysis was conducted using dataset of disease incidence as dependent variable and climatic factors as explanatory variables obtained from the four-year exposure tests. The results showed that disease incidence was strongly correlated with a total number of two consecutive rainy days. Correlation coefficient (R**2) was 0.366 in June-July (Y1) and 0.640 in August-September (Y2). (3) Latent period inferred from the dataset was ca. 14 days in 2002 and 20-38 days in 2003 on assumption that infection occurred on the first rainy day. V Susceptability of G. triflora and G. scabra to M. gentianae 1. Inoculation tests for seedlings showed that M. gentianae strain K18 (isolated from G. triflora) and MAFF 239231 (isolated from G. scabra) caused lesions on all six G. triflora cultivars but failed to infect G. scabra cv. Aruta. 2. Inoculation tests for cut plants showed that M. gentianae strain K18 caused lesions on all ten G. triflora cultivars, as well as cv. Lovely-ashiro and cv. New-hybrid-ashiro (both origins unknown), but failed to infect two G. scabra cultivars (cv. Ashiro-no-sawakaze and cv. Aruta) and an interspecific hybrid between G. triflora and G. scabra, cv. Arubireo. 3. Parents of the interspecific hybrid cv. Arubireo, G. triflora cv. Ba was susceptible but G. scabra cv. OK was resistant for the causal fungus. These results indicate that the resistance of G. scabra is inherited dominantly. VI Disease control 1. Fungicide application tests in gentian field showed that basic copper sulfate, TPN, and kresoxym-methyl were highly effective as reported in the previous study, and the present study revealed the effectiveness of thiuram. 2. The relationship between TPN-application timing and the control efficacy was evaluated for three year times to examine infection periods in the field. The results showed that any of the application between late June and late July could control the disease with two consecutive applications. In 2002 and 2003, the application around early July was effective, which agreed with the fact that inoculum potential culminates in early July. Four consecutive application from late June to late July with 10 day intervals was superior to two consecutive applications around early July, in that the latter method failed to control disease development until October, despite of controlling perfectly until August. In conclusion, fungicide application for the disease control should be made consecutively in late June-late July (the rainy season) in Iwate. VII The life cycle of the causal fungus and disease ecology in Iwate Results revealed that the brown leaf spot fungus on gentian was anamorphic in the field. Life cycle of the fungus and disease epidemiology in Iwate are summarized as follows. 1. Life cycles A primary inoculum source is conidia formed on overwintered, infected leaves in the rainy season (late June to late July). Minute lesions appear sporadically on leaves in early August. The diseased leaves become secondary inoculum source and form conidia (sporophores) on the lesions. The conidia are dispersed into rain drops, which splash the conidia widely. The causal fungus overwinters as the hyphal mat under leaf epidermal cells. Overwintered, infected leaves serve as the primary inoculum source the following year. 2. The primary inoculum source Overwintered, infected leaves as the primary infection source, form sporophores under the weather conditions in which temperature reaches about 15 degC and leaf wetness duration continues for several days. In Iwate, primary infection continues between late June and late July, and infection frequency is higher around early July, i.e., just before the rainy season. 3. Infection and disease development Leaf wetness duration is a limiting factor for conidial infection. Long period of leaf wetness (more than 36 h) are required as compared to other general plant pathogens. Temperature for conidial infection ranges from 15 to 25 degC, and higher temperature allows rapid infection within short duration of leaf wetness. Germinated conidia form appressoria on the surface of gentian leaves. Appressorium formation is promoted by high temperature and long leaf wetness duration. Appressorium may form infection hypha for invasion under epidermis of the leaves. Further studies still remain to clarify an invasion mode. Latent period for disease development is affected by temperatures. At 25 degC, lesions obviously develop ca. 14 d after inoculation, although the period tends to extend at lower temperatures. High temperatures tend to stimulate disease development rapidly, which agrees with field observations that lesions usually appear in early August. The appearance may be promoted by rapidly increase in temperature after the end of the rainy season. 4. Secondary inoculum source Sporophores are formed on diseased leaves when the lesion tissues turn brown and rotten and when humidity reaches > 99% RH (the optimum 100% RH). Long duration of leaf wetness leads to an increase in sporophore formation. Conidia produced on diseased leaves are dispersed into rain drops, and the droplet splashes and helps the pathogen infect leaves. Primary lesions appearing in the fields in early August, even though still sporadic, could provide sufficient inoculum in the late phase of epidemics.