Apple White Rot Research Articles

Biological Control of White Rot in Apple Using Bacillus spp.

Apple white rot, caused by <i>Botryosphaeria dothidea</i>, is one of the important diseases in Korea. <i>B. dothidea</i> can cause pre- and postharvest decay on apple fruit as well as canker and dieback of apple trees. In this study, we isolated bacteria from the trunk of apple trees and tested their antagonistic activity against <i>B. dothidea</i>. Five bacterial isolates (23-168, 23-169, 23-170, 23-172, and 23-173) were selected that were most effective at inhibiting the mycelial growth of the pathogens. The isolate 23-172 was identified as <i>Bacillus amyloliquefaciens</i> and four isolates 23-168, 23-169, 23-170, and 23-173 were identified as <i>Bacillus velezensis</i> by RNA polymerase beta subunit (<i>rpoB</i>) and DNA gyraseA subunit (<i>gyrA</i>) gene sequencing. All isolates showed strong antagonistic activity against <i>B. dothidea</i> as well as <i>Colletotrichum fructicola</i> and <i>Diaporthe eres</i>. All isolates exhibited cellulolytic, proteolytic and phosphate solubilizing activities. In particular, two isolates 23-168, 23-169 were shown to significantly reduce the size of white rot lesions in pretreated apple fruits. These results will provide the basis for the development of a fungicide alternative for the control of white rot of apple.

An efficient gene disruption method for the woody plant pathogen Botryosphaeria dothidea

BackgroundBotryosphaeria dothidea causes apple white rot and infects many tree plants. Genome data for B. dothidea are available and many pathogenesis-related genes have been predicted. However, a gene manipulation method is needed to study the pathogenic mechanism of B. dothidea.ResultsWe established a gene disruption (GD) method based on gene homologous recombination (GHR) for B. dothidea using polyethylene glycol-mediated protoplast transformation. The results showed that a GHR cassette gave much higher GD efficiency than a GHR plasmid. A high GD efficiency (1.3 ± 0.14 per 106 protopasts) and low frequency of random insertions were achieved with a DNA cassette quantity of 15 μg per 106 protoplasts. Moreover, we successfully disrupted genes in two strains. Bdo_05381-disrupted transformants produced less melanin, whereas the Bdo_02540-disrupted transformant showed a slower growth rate and a stronger resistance to Congo red.ConclusionThe established GD method is efficient and convenient and has potential for studying gene functions and the pathogenic mechanisms of B. dothidea and other coenocytic fungi.

Identification of Differentially Up-regulated Genes in Apple with White Rot Disease.

Fuji, a major apple cultivar in Korea, is susceptible to white rot. Apple white rot disease appears on the stem and fruit; the development of which deteriorates fruit quality, resulting in decreases in farmers' income. Thus, it is necessary to characterize molecular markers related to apple white rot resistance. In this study, we screened for differentially expressed genes between uninfected apple fruits and those infected with Botryosphaeria dothidea, the fungal pathogen that causes white rot. Antimicrobial tests suggest that a gene expression involved in the synthesis of the substance inhibiting the growth of B. dothidea in apples was induced by pathogen infection. We identified seven transcripts induced by the infection. The seven transcripts were homologous to genes encoding a flavonoid glucosyltransferase, a metallothionein-like protein, a senescence-induced protein, a chitinase, a wound-induced protein, and proteins of unknown function. These genes have functions related to responses to environmental stresses, including pathogen infections. Our results can be useful for the development of molecular markers for early detection of the disease or for use in breeding white rotresistant cultivars.

Identification of a Novel Hypovirulence-Inducing Hypovirus From Alternaria alternata.

Mycoviruses are wide spread throughout almost all groups of fungi but only a small number of mycoviruses can attenuate the growth and virulence of their fungal hosts. Alternaria alternata is an ascomycete fungus that causes leaf spot diseases on various crop plants. In this study, we identified a novel ssRNA mycovirus infecting an A. alternata f. sp. mali strain isolated from an apple orchard in China. Sequence analyses revealed that this virus is related to hypoviruses, in particular to Wuhan insect virus 14, an unclassified hypovirus identified from insect meta-transcriptomics, as well as other hypoviruses belonging to the genus Hypovirus, and therefore this virus is designed as Alternaria alternata hypovirus 1 (AaHV1). The genome of AaHV1 contains a single large open-reading frame encoding a putative polyprotein (∼479 kDa) with a cysteine proteinase-like and replication-associated domains. Curing AaHV1 from the fungal host strain indicated that the virus is responsible for the slow growth and reduced virulence of the host. AaHV1 defective RNA (D-RNA) with internal deletions emerging during fungal subcultures but the presence of D-RNA does not affect AaHV1 accumulation and pathogenicities. Moreover, AaHV1 could replicate and confer hypovirulence in Botryosphaeria dothidea, a fungal pathogen of apple white rot disease. This finding could facilitate better understanding of A. alternata pathogenicity and is relevant for development of biocontrol methods of fungal diseases.

Characterization of a novel strain of Botryosphaeria dothidea chrysovirus 1 from the apple white rot pathogen Botryosphaeria dothidea.

A novel strain of Botryosphaeria dothidea chrysovirus 1 was identified. It encodes a shortened RNA-dependent RNA polymerase and an elongated coat protein, and it might cause hypovirulence of the host fungal strain.

Sensitivity of Botryosphaeria dothidea from apple to tebuconazole in China

The white rot disease caused by Botryosphaeria dothidea threatens apple production in Bohai bay area and along the Yellow River of China, where disease control is largely dependent on fungicides such as tebuconazole. A total of 146 isolates of B. dothidea obtained from different apple orchards in six provinces were tested for their sensitivity to tebuconazole, carbendazim and iprodione. The EC50 values of all tested isolates for tebuconazole were from 0.035 to 1.415 μg/mL. The broad range of EC50 values of tebuconazole suggests an obvious variation among the 146 isolates. Isolate HB13 (EC50 = 1.415 μg/mL) showed reduced sensitivity to tebuconazole, with an EC50 value significantly higher than those of the other 145 isolates tested. The low sensitivity of HB13 was stable after 15 generations, and this isolate showed similar pathogenicity as susceptible strains. EC50 correlation analysis indicates no cross resistance between tebuconazole and carbendazim and iprodione. Field efficacy trials showed that tebuconazole remains very effective for apple white rot control in China.

Effects of Interrupted Wetness Periods on Conidial Germination, Germ Tube Elongation and Infection Periods of Botryosphaeria dothidea Causing Apple White Rot.

Responses of Botryosphaeria dothidea to interrupted wetness periods were investigated under in vivo and in vitro conditions. Conidia of B. dothidea were allowed to germinate on apple fruits under wetting condition at 25ºC for 5 hr. They were air-dried for 0, 1, 2 or 4 hr, and then rewetted at 25ºC for 5 hr. Following an initial wetness period of 5 hr, 83% of the conidia germinated. The percent conidial germination increased to 96% when wetting was extended continuously another 5 hr. However, no further conidial germination was observed when wetting was interrupted by dry periods of 1, 2 and 4 hr, resulting in 83, 81 and 82%, respectively. The mean length of the germ tubes was 37 μm after 5 hr of wetting and elongated to 157 μm after 10 hr of continuous wetting. On the other hand, interruption of wetting by a dry period of 1 hr or longer after the 5 hr of initial wetting arrested the germ tube elongation at approximately 42 μm long. Prolonged rewetting up to 40 hr did not restore germ tube elongation on slide glasses under substrate treatments. Model simulation using weather data sets revealed that ending infection periods by a dry period of at least 1 hr decreased the daily infection periods, avoiding the overestimation of infection warning. This information can be incorpo- rated into infection models for scheduling fungicide sprays to control apple white rot with fewer fungicide applications.

After-infection Activity of Protective Fungicides against Apple White Rot

In a trial to select suitable fungicides for developing a spray program that can control apple white rot effectively, after-infection activities in some protective fungicides were detected. Six fungicides, mancozeb, propineb, benomyl, folpet, azoxystrobin and iminoctadine-triacetate, which had been extensively used in apple orchards, were sprayed on 12-year-old apple trees (cv. Fuji) at 15-day intervals from late May to late July. Disease incidences and infection frequencies of the fruit bagged just before and soon after each spray were examined. When the infection frequency or disease incidence of the fruit bagged after each spraying of fungicide was significantly lower than those of the fruit bagged before spraying, the fungicides appeared to confer after-infection activity. The six fungicides showed diverse activities on white rot: folpet showed after-infection activity on disease development, iminoctadine-triacetate showed after-infection activity on infection, azoxystrobin showed after-infection activity on disease development and infection, and mancozeb, propineb and benomyl showed no distinct activity. The activity of a fungicide became much higher when it was sprayed alternately with other fungicide rather than successive spraying of the same fungicide. Analysis of the properties of these protective fungicides could lead to the development of a highly effective spray program against white rot.

An Infection Model of Apple White Rot Based on Conidial Germination and Appressorium Formation of Botryosphaeria dothidea

Regression models for determining infection periods of apple white rot were developed based on conidial germination and appressorium formation of Botryosphaeria dothidea. A total of 120 apple fruits were inoculated with the fungal conidial suspension and subjected to 6 temperatures and 10 wetness periods. Conidia germinated and produced appressoria, exhibiting swollen tips of germ tubes on the fruit surface. Conidial germination (G) increased with temperature (T) and wetness period (W), and was described as <TEX>$G=-89.273+7.649T+7.056W-0.109T^{2}-0.085W^{2}-0.066TW(R^{2}=0.75)$</TEX>. Less than 2 hr of wetness period were enough for conidia to germinate at 25 to <TEX>$30^{\circ}C$</TEX>. Effects of temperature and wetness period on appressorium formation (A) could be explained as <TEX>$A=-1.540-2.375W+0.045W^{2}+0.213TW(R^{2}=0.77)$</TEX>. The relationship between conidial germination and appressorium formation (<TEX>$A_g$</TEX>) was described as<TEX>$A_g=0.381-0.227G+0.005G^{2}(R^{2}=0.67)$</TEX>, suggesting that conidial germination may have to reach approximately <TEX>$43.7\%$</TEX> to initiate appressorium formation. Using the regression equation for conidial germination and the criterion of <TEX>$43.7\%$</TEX> conidial germination, an infection model was developed to determine infection periods based on temperature and wetness period. The infection model with the criterion of <TEX>$43.7\%$</TEX> conidial germination was apparently more conservative than the appressorium formation model in determining possibility of apple infection. The infection model seemed sensitive to variable weather conditions, suggesting possible use of the model for timing fungicide sprays to control white rot of apples in practice.

Effect of Application Timing of Ergosterol Biosynthesis-inhibiting Fungicides on the Suppression of Disease and Latent Infection of Apple White Rot Caused by Botryosphaeria dothidea

To examine the duration of post-infectional activity of ergosterol biosynthesis-inhibiting fungicides (EBIs) against Botryosphaeria dothidea, the cause of apple white rot, a series of experiments was conducted for 3 years at different times during the apple-growing season using apples infected through artificial inoculation or natural infection. Disease incidence at harvest was gradually reduced as the time of chemical application was delayed until early or mid-August, regardless of the time of infection. Latent infections, detected after storing the fruit at 25°C for 4 weeks were also reduced by late season applications, but the reduction was less than that observed on the fruit at harvest. From these results, apple white rot can be effectively controlled by a single application of EBIs around mid-August, after which time, infections no longer occur in Korea.

Latency- and Defense-Related Ultrastructural Characteristics of Apple Fruit Tissues Infected with Botryosphaeria dothidea

ABSTRACT Apple fruit tissues infected with Botryosphaeria dothidea were examined by transmission electron microscopy using susceptible cv. Fuji and resistant cv. Jonathan. Immature (green) and mature (red) fruits of cv. Fuji with restricted or expanding lesions were also examined to reveal subcellular characteristics related with latent and restricted disease development. In infected susceptible mature fruits, cytoplasmic degeneration and organelle disruption commonly occurred, accompanying cell wall dissolution around invading hyphae. Cell wall dissolution around invading hyphae in subepidermis was rare in immature, red halo-symptomed cv. Fuji and resistant cv. Jonathan fruits. In infected immature fruits of cv. Fuji, presumably at the latent state of disease development, cellular degeneration was less severe, and invading hyphae contained prominent microbody-lipid globule complexes or the deposition of thin electron-dense outer layer around cell wall of intercellular hyphae. Both mature fruits with red halos and resistant apple fruits formed cell wall protuberances at the outside of cell walls. In addition, electron-dense extramural layers were formed in the resistant apple fruits. Aberrant hyphal structures such as intrahyphal hyphae were found only in resistant fruit tissues, indicating the physiologically altered fungal growth. These ultrastructural changes of host tissues and fungal hyphae may reflect the pathogenesis of apple white rot under varying conditions of apple fruits.