Effects of panicle development stage and temperature on rice panicle blast infection by Magnaporthe oryzae and visualization of its infection process

Panicle blast, caused by the fungus Magnaporthe oryzae (syn. Pyricularia oryzae), directly contributes to yield loss in the field. The effects of panicle development stage and temperature on panicle blast were studied and the infection process of M. oryzae in panicles was visualized. Rice panicles at different development stages from three rice cultivars were inoculated with a conidial suspension in vitro. The rice cultivar Lijiangxintuanheigu was highly susceptible to panicle blast at 5 days postinoculation (dpi) when the pulvinus distance was 15–20 cm. Nanjing 9108 was moderately susceptible to panicle blast when the pulvinus distance was 8–10 cm, but Yliangyou 800 was resistant. The effect of temperature on panicle blast was determined under 22–35 °C temperature treatments. Inoculated panicles placed at temperatures of 28 and 30 °C showed the highest lesion grade based on lesion length at 5 dpi. The infection process of M. oryzae in rice panicles was observed by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). M. oryzae initially formed the appressorium to invade through the epidermis of rice panicles at 24 hours postinoculation (hpi). As the disease progressed, the invasive hyphae formed dense mycelial networks in the inner parenchyma cells at 60 hpi. Our results will contribute to the understanding of panicle development stage and temperature effects on panicle blast and improve resistance evaluation methods. Additionally, visualization of the infection process by CLSM and TEM are valuable methods to observe M. oryzae invasive hyphae inside rice panicle cells.     

Long‐term survival of blast pathogen in infected rice residues as major source of primary inoculum in high altitude upland ecology

Magnaporthe oryzae is the fungal plant pathogen that causes rice blast. The sources of primary inoculum and overwintering mode of the fungus remain largely unknown. The effect of rice residues on the onset of blast epidemics and the potential for survival of M. oryzae in the residues were studied in upland conditions in Madagascar. Blast disease was observed in a 3‐year field experiment in three treatments: with either infected or uninfected rice residues on the soil surface, or without rice residues. Leaf blast incidence was significantly higher in the treatment with infected rice residues than in the two other treatments at the early stages of the epidemic. In a second set of trials, the survival of M. oryzae on rice residues was monitored. Infected rice stems were placed by lots in three places: on the mulch of rice residues, under the mulch, and buried at a depth of 10 cm in the soil. Each month, samples were taken from the field and tested for sporulation. The survival of the blast fungus decreased rapidly on the stems buried in the soil but remained high for the other conditions. Sporulation of the fungus was observed on stems left on the mulch for up to 18 months. It is concluded that under field conditions, the presence of infected rice residues could initiate an epidemic of blast. The results of this study may help in designing effective management strategies for rice residues infected by M. oryzae.     

Magnaporthe oryzae conidia on basal wheat leaves as a potential source of wheat blast inoculum

Wheat blast caused by Magnaporthe oryzae Triticum causes significant losses on wheat during outbreak years in several South American countries. Despite reports of wheat blast leaf lesions on some wheat cultivars, the importance of inoculum originating from leaves in severely affected commercial fields is disputed. It is generally considered that leaf lesions and/or sporulation on leaves do not usually appear before the occurrence of spike blast in wheat. The purpose of this study was to (i) determine the occurrence of wheat blast on basal leaves, (ii) estimate the number of conidia produced on these leaves, and (iii) determine the impact of current fungicide application practices on inoculum produced from sporulating lesions on basal wheat leaves. Inoculations at the three‐leaf stage showed that certain cultivar and isolate combinations caused more disease on old wheat leaves than young expanding leaves. Under optimum conditions, M. oryzae had the potential to produce tens to hundreds of thousands of conidia on small amounts of wheat basal leaves. A mean of 1 669 000 conidia were produced on 1 g dry basal leaves of a highly susceptible cultivar under optimum conditions for sporulation. Conidia production on leaves coincided with spike emergence under both greenhouse and field conditions. When field studies were conducted under natural epidemic conditions, foliar fungicide applications reduced the amount of M. oryzae conidia on basal leaves by 62–77% compared to non‐sprayed controls. An earlier application of foliar fungicides might reduce inoculum if conidia from basal leaves contribute to wheat spike blast development.     

Suppression of Rice Blast by Preinoculation with Avirulent Pyricularia oryzae and the Nonrice Pathogen Bipolaris sorokiniana

ABSTRACT Avirulent isolates of Pyricularia oryzae and isolates of Bipolaris sorokiniana, a nonrice pathogen, were used to suppress rice blast caused by P. oryzae. In greenhouse experiments, both fungi substantially reduced leaf blast when applied 24 h or more before the pathogen. B. sorokiniana, but not avirulent isolates of P. oryzae, systemically reduced disease in leaf 5 when applied to whole plants at the four-leaf stage. In field experiments, both fungi were able to reduce neck blast significantly. No increase in grain yield was obtained by using avirulent isolates of P. oryzae, whereas five sprays with B. sorokiniana from seedling to heading stages increased the grain yield in two of three experiments conducted at two locations in Nepal. The significant increase in yield was observed under high inoculum pressure of P. oryzae. Induced resistance is suggested to be involved in the suppression of disease.     

Infection processes of Ustilaginoidea virens during artificial inoculation of rice panicles

Ustilaginoidea virens is a ubiquitous plant pathogen that causes rice false smut disease, one of the most destructive diseases of rice (Oryza sativa L.) production. However, data concerning the effect of inoculation on disease development and the infection process of this pathogen are not comprehensive. In this study, the developmental processes of U. virens in rice panicles were characterized using an enhanced green fluorescent protein (EGFP) labelled strain. A mixture of hyphae and conidia of U. virens was used to inoculate rice panicles by leaf sheath injection during the booting stage of rice plants grown in a greenhouse. The panicles were assessed to determine the relationship between artificial inoculation and disease occurrence. Increasing volumes of inocula (0.2, 0.5, 1, and 2 ml of a mixture of hyphae-fragment and 2?×?10⁶ conidia/ml suspension) caused more severe infections, and small differences were also observed for the different inoculation sites at the base, apex and mid-point of rice panicles. The optimum inoculation condition was 1–2 ml inoculum injected into the mid-point of rice panicles. Spikelet samples were collected as the disease progressed and observed by confocal laser scanning microscopy and scanning electron microscopy. The images collected showed that the primary site of U. virens colonization was at the base of the filaments with the inner spikelets becoming infected by hyphae at 24 h post inoculation (hpi). The accumulation of hyphae reached its highest level at 168 hpi, before the rice heading stage, as the infection extended upward from basal filaments to the anther apex, and then enclosed all the floral organs to produce a velvety smut ball.     

水稻凋萎型白叶枯病稻桩传病规律的研究

通过接种,小区试验和大田验证,证明凋萎型白叶枯病稻桩上潜在病菌可以存续至第二年5月份后并仍能传病,因之它是稻草、种子以外的又一主要菌源。初步看来稻桩上的病菌主要在秧田期传染,但菌量大时,也可以在本田期侵染,所以在凋萎型白叶枯病严重地区,除狠抓秧田期防治外,还应当注意本田前期的防治。     

Overwintering of <Emphasis Type="Italic">Pyricularia oryzae</Emphasis> in wild infected foxtails

The rice blast fungus Pyricularia oryzae mainly overwinters in infested rice organs stored indoors, whereas it is difficult or impossible for the pathogen to overwinter outdoors. By contrast, blast pathogens infecting weed grasses must overwinter outdoors every winter to continue their life cycle. In this study, we investigated the overwintering location of P. oryzae infecting wild, green, and giant foxtails to identify the mechanism that enables them to overwinter. Recovery of P. oryzae was tested in seeds of wild foxtail collected from the soil surface from December to April over three winters. No P. oryzae was recovered from the seed samples of any wild foxtail collected at the ends of the three experimental periods in April. Recovery was also tested from blast lesions on leaves and seeds sampled from withered green foxtail in the experimental field of Saga University from November to April during two winters. In contrast to seeds on the soil surface, P. oryzae survived in lesions and seeds at the ends of the two experimental periods during April, suggesting that withered host plants could be the overwintering site of the pathogen. Rice plants are reaped and removed from paddy fields after harvesting. Thus, withered, standing plants may be available solely to blast pathogens infecting wild grasses, possibly explaining the higher winter survival frequency of weed pathogens than that of rice blast pathogens outdoors.     

Colonization of the vegetative stage of rice plants by the false smut fungus Villosiclava virens,as revealed by a combination of species‐specific detection methods

Rice false smut disease caused by the ascomycete fungus Villosiclava virens (Clavicipitaceae) reduces rice yield worldwide. It invades rice panicles and forms dark‐green false smut balls composed of thick‐walled conidia. Although the infection process during the booting stage is well studied, its infection route before this is unclear. It was hypothesized that the thick‐walled conidia in soil penetrate rice roots, and the fungus latently colonizes roots and tiller buds at the vegetative stage. This hypothesis was tested using species‐specific detection methods. First, real‐time PCR with species‐specific primers and probe was used to estimate thick‐walled conidial number in the paddy field soil. Secondly, nested PCR with species‐specific primers showed that fungal DNA was detected in roots and shoot apices of rice plants in the vegetative stage. Thirdly, colourimetric in situ hybridization with a species‐specific oligonucleotide probe targeting 18S rRNA suggested that sparse mycelia or tightly condensed mycelia were present on the external surface of tiller buds enveloped by juvenile leaf sheaths at the vegetative stage. Thin hyphae were found around leaf axils at the surface of elongated stems at the heading stage, and the fungal hyphae grew in the rice root tissues. In addition, it was demonstrated that eGFP‐tagged transformants of the fungus invaded rice roots and colonized the surface of roots and leaf sheaths under artificial conditions.     

Resistance in leaf blades assessed by counting conidia correlates with whole-plant-specific resistance in leaf sheaths in a compatible rice–Magnaporthe oryzae interaction

Resistance in the leaf blades of rice plants against a virulent race of the rice blast fungus Magnaporthe oryzae was quantitatively examined using a modified spot inoculation method. Numbers of conidia produced in the lesions were affected by plant age and paralleled the frequency of resistance infection types, which is indicative of whole-plant-specific resistance (WPSR), in the inoculated leaf sheaths of the corresponding plants. Exogenous abscisic acid treatment, which suppresses WPSR, also increased the susceptibility of the leaf blades. These results indicate a correlation between the resistance of the leaf blades and the WPSR in the leaf sheaths.     

Management of bakanae and bacterial seedling blight diseases in nurseries by irradiating rice seeds with atmospheric plasma

The control of seedborne rice seedling diseases in the seed beds is important to avoid epidemics in rice nurseries and paddies, which may result in severe yield loss. Recently, irradiation with plasma containing electrons, creating positive or negative ions and neutral species, has been shown to have an antimicrobial effect, probably via generation of reactive oxygen species. This study examines whether two seedborne rice seedling diseases, bakanae disease caused by the fungal pathogen Fusarium fujikuroi, and bacterial seedling blight caused by Burkholderia plantarii, are suppressed by irradiation of infected rice seeds with atmospheric plasma. Seed germination and seedling growth were not inhibited in plasma‐treated healthy seeds. When F. fujikuroi‐infected rice seeds were irradiated with plasma after being immersed in sterile distilled water, bakanae disease severity index and the percentage of plants with symptoms were reduced to 18.1% and 7.8% of non‐irradiated control, respectively, depending on the duration of plasma irradiation. The bacterial seedling blight disease index was also reduced by plasma irradiation in vacuum‐inoculated seeds to 38.6% of the non‐irradiated control, and in infected seeds harvested from spray‐inoculated heads of rice plants to 40.1% of the control. Therefore, plasma irradiation seems to be effective in controlling two independent seedborne rice seedling diseases.     

Expression patterns of defence genes and antioxidant defence responses in a rice variety that is resistant to leaf blast but susceptible to neck blast

The rice stage resistance variety Jiajing3768 is resistant to leaf blast but susceptible to neck blast. This variety was used to analyze the expression patterns of defence genes and antioxidant defence responses in the leaves at the seedling stage (LSS) and necks at the preliminary heading stage (NPHS), after inoculation with Magnaporthe oryzae. We found that defence genes PR1b (pathogenesis-related class 1b), PR4, PR10a, JIOsPR10 (jasmonic acid induced rice PR 10), Gns1 (1,3; 1,4-β-glucanase), Cht-1 (chitinase), and LOX (lipoxygenase) may play roles in the stage resistance in Jiajing3768; PAL (phenylalanine ammonia-lyase) and CHS (chalcone synthase) could participate in defending Jiajing3768 against neck blast but not leaf blast. The antioxidant enzymes superoxide dismutase, peroxidase, catalase, glutathione reductase, and malondialdehyde coordinately participate in the stage resistance to blast in Jiajing3768, and that oxidative damage is less in the LSS than in the NPHS.     

Transgenic rice plants that over-express the mannose-binding rice lectin have enhanced resistance to rice blast

Mannose-binding rice lectin (MRL), which is almost identical to the salt-induced protein SalT, binds to mannose and glucose residues. Expression of the MRL gene in response to infection with Magnaporthe oryzae, the rice blast fungus, was stronger in the incompatible interaction than in the compatible. Transgenic rice plants that constitutively over-expressed MRL strongly suppressed the growth of invasive hyphae of the fungus on leaf sheaths and the development of typical susceptible-type lesions on leaf blades, but did not affect penetration by the fungus in comparison with the wild-type. On a polycarbonate plate, purified recombinant MRL inhibited conidial attachment and appressorium formation but not conidial germination. These results suggest that MRL may play an essential role in disease resistance by suppressing development of M. oryzae in situ.     

Management of rice blast (Pyricularia oryzae): implications of alternative hosts

Rice blast (Pyricularia oryzae) has become a serious disease on commercial rice (Oryza sativa) crops in northern Australia and is present there on wild rice (O. australiensis). Characterisation of the host range of P. oryzae is fundamental to both reducing disease spread and to preventing development of epidemics via better management of Poaceae inoculum reservoirs in Australia. Studies on response of three different wild O. australiensis sources toward four isolates of P. oryzae showed all genotypes very susceptible to three isolates [WAC13466 (race IA-1), BRIP53376 (race IB-3), NT2014a (race unknown)], but resistant to isolate BRIP39772 (race IA-3). Studies to investigate levels of blast disease development following inoculation on a range of Poaceae hosts showed both P. oryzae isolates (WAC13466, BRIP53376) were highly virulent on barley (disease index, DI?=?100%), and on Phalaris and O. australiensis (DI?=?70%). However, isolate BRIP53376 showed a significantly higher level of aggressiveness (DI ~80%) on ryegrass, wild oat and rice. Of the two wheat cultivars tested, only one cultivar showed disease and only from WAC13466 (DI ~30%). Sweet corn and goosegrass were also susceptible to both blast isolates, but DI was <50%. That P. oryzae was virulent across these diverse Poaceae hosts, highlights, for Australia, the possibility for these species in, first, harbouring P. oryzae isolates highly virulent to commercial rice, second, fostering spread of rice-attacking P. oryzae strains into regions currently free of rice blast, and third, potential for these alternative host species to encourage development of more virulent host-specific strains of P. oryzae. The current study is an important step towards facilitating improved crop protection in the medium and long term from reducing P. oryzae disease epidemics via a better understanding and management of inoculum reservoirs in Australia.

     

Effects of leaf blast on photosynthesis of rice 2. Canopy photosynthesis

The effect ofPyricularia oryzae, the causal organism of leaf blast in rice, on photosynthetic rate of a rice crop was determined with mobile equipment in the field. Canopy CO₂ exchanges rate (CER) was significantly reduced in plots inoculated withP. oryzae. The experimentally obtained data were used to evaluate the performance of a model for the effects of leaf blast on canopy photosynthesis. The model comprised photosynthesis and respiration routines of a mechanistic crop growth model, extended with submodels for effects of leaf blast on both processes. Canopy photosynthesis and the effect of leaf blast on CER were accurately simulated with the model.Analysis showed that the reduction in canopy photosynthesis was mainly due to an adverse effect of lesions on leaf photosynthetic rate and to shading by dead leaf are resulting from disease induced senescence. A sensitivity analysis demonstrated the importance of the vertical distribution of the disease in the canopy. This implies that disease monitoring for crop loss assessment should consider vertical disease distribution.     

Epiphytic colonization of Ustilaginoidea virens on biotic and abiotic surfaces implies the widespread presence of primary inoculum for rice false smut disease

Ustilaginoidea virens (Uv), the causative agent of rice false smut disease, infects developing rice spikelets at the booting stage, and transforms individual grains of the panicle into smut balls. Epidemics of the disease occur when the rice booting and heading stages coincide with rainy days. Using a green fluorescent protein (GFP)‐labelled Uv isolate that can form false smut balls on rice panicles, it was found that under high humidity and free water conditions the Uv isolate could colonize leaves of plants belonging to various families including the Poaceae (Oryza sativa, Echinochloa crusgalli, Digitaria sanguinalis and Leptochloa chinensis), the Brassicaceae (Arabidopsis thaliana) and the Solanaceae (Nicotiana benthamiana) without symptoms. Over several days, some conidia could germinate on the leaves of these plants and in water on the surface of Parafilm and cellophane, form hyphae and differentiate conidiophores to generate a large number of secondary conidia, while other conidia were able to directly produce secondary conidia. Conversely, in the absence of water some conidia could either bud to form new conidia or were converted into chlamydospores. These data indicate that Uv is one of a few fungal pathogens reported to have epiphytic characteristics. The rapid generation of a large number of spores on biotic and abiotic surfaces greatly increases the inoculum that can infect rice spikelets, resulting in the occurrence of rice false smut disease epidemics. These findings are important in the development of disease control strategies.     

Response of the panicles exserted from the caulis and from various effective tillers at four stages of panicle development to neck blast in rice

The rice japonica variety Nipponbare and the indica variety 93-11, the genomic DNA sequences of which are known, were used to analyze the response of the panicles exserted from the caulis and from various effective tillers at four stages of panicle development to neck blast. Disease incidence in the necks (DIN), disease incidence in the rachis nodes (DIRN), lesion length in the necks (LLN), and number of conidia in the necks (NCN) were measured after inoculating the panicles in vitro of two rice varieties with Magnaporthe oryzae. Both Nipponbare and 93-11 were susceptible, DIN and DIRN of all panicles being 100% in both the varieties except DIRN in several panicles at stage 1 (the panicle fully exserted) in Nipponbare. Both LLN and NCN of panicles decreased as the panicles continued to develop. However, the patterns of this decrease in the panicles from the caulis and from various effective tillers were substantially different in the two varieties. In Nipponbare, neck blast became progressively severe in the order of emergence of the panicles from the caulis and from six effective tillers: values of LLN and NCN were the lowest in the panicles from the caulis, intermediate in those from first-class tillers, and the highest in those from second-class tillers. In 93-11, however, the source of panicles had no significant influence on LLN and NCN.     

三环唑浸秧防治稻叶瘟技术研究

用20％三环唑(tricyclazole)可湿性粉剂750—1000倍液浸湿秧苗半分钟左右取出,堆置半小时后移栽,药后33—55天防效达85％以上,本田不再施药即可有效地控制本田叶瘟。较常规喷雾更有效地发挥了三环唑内吸性强、持效期长、耐雨水冲刷的特点,减少本田施药1—2次,省工省药且方法简便易行。通过四川大面积示范推广,效益非常显著,为稻瘟病综合防治提供了一项有效措施。     

Effect of carpropamid on secondary infection by rice blast fungus

Carpropamid (WIN™, KTU 3616) provides good control of leaf and panicle blast by ‘one-shot’ nursery-box treatment. It inhibits melanin biosynthesis in appressorial cells of Pyricularia oryzae, making them hyaline. Penetration by infection hyphae from the hyaline appressoria into rice epidermal cells is substantially hindered. In addition, the spread of rice blast spores from primary lesions to the other parts of the plant leading to secondary infection is largely prevented when the plants are treated with carpropamid by spray or water surface application. Secondary infection was simulated in a glass chamber fitted with an ultrasonic humidifier. On treated plants, many blast spores formed in the lesions, but the number of air spora that were dispersed from the lesions decreased significantly. A similar suppression of the spore liberation was observed in vitro when lesions on rice leaf segments, or discs from Pyricularia cultures on oatmeal agar were treated with the chemical. Spores from treated lesions or from the cultures on oatmeal agar amended with the chemical germinated normally and produced well-melanized appressoria on cellophane membranes. In addition, the spores proved to be fully pathogenic towards rice seedlings, producing normal disease symptoms. These results strongly suggest that carpropamid reduces the secondary infection of rice by Pyricularia by specifically hindering spore liberation. © 1999 Society of Chemical Industry     

Silicon content in rice seedlings to protect rice blast fungus at the nursery stage

To control rice blast effectively at the nursery stage, the absolute SiO₂ content necessary for rice plants to resist blast disease was investigated using various rice cultivars and soils. Nine rice cultivars with different complete resistance genes and different degrees of partial resistance were grown on nursery soils amended with silica gel at different rates to change the SiO₂ content of rice plant. The rice seedlings were then inoculated 28 days after sowing with Pyricularia grisea to estimate their blast resistance. In all rice cultivars, the number of lesions was significantly reduced when SiO₂ content increased in the rice seedling; lesions were reduced to 5%–20% of the number on the seedlings grown in soil without silica gel when the seedling SiO₂ content reached 5%. Additionally, the susceptibility to blast disease of rice seedlings grown on eight soils collected from different districts, with varying amounts of silica gel, was compared. The number of lesions decreased significantly when the SiO₂ content in the seedlings reached 5%. These results suggest that SiO₂ content of at least 5% in the rice plant can control this disease at the nursery stage under any conditions.