Identification Of The Pathogen Causing Adlay Anthracnose And Study On The Role Of Nitric Oxide In Plant Disease Resistance

Anthracnose caused by Colletotrichum species is a type of generally occurring and economically important plant disease. Research on plant resistance to this disease will provide some basis on control of this disease. Among the manifesting types of plant disease resistance, nonhost resistance and resistance gene-dependent gene-for gene resistance are two important ones. This thesis research attempts to identify a new disease of adlay (Coix lacryma-jobi var. ma-yuen Stapf)-adlay anthracnose;analyse nonhost resistance of rice to Colletotrichum gloeosporioides;and study the role of nitric oxide (NO) in nonhost resistance and gene-for gene resistance. The main results are as follows:1. A new adlay disease was characterized and identified. The disease occurred with the following symptoms: after infection, leaves and stems showed small water-soaked brownish spots which rapidly turned into longitudinal elliptic, or spindle-shaped lesions 6-8 x 2-4 cm in size, with a brown outer edge and a gray sunken central area. Coalescence of large lesions gave rise to extensive rotting and necrosis that, when stems were encircled, caused them to break, thus inducing significant yield losses. Through fulfillment of the Koch's postulation by microorganism isolation from disease sample, adlay plant re-inoculation with the isolates, and microorganism re-isolation from the re-inoculated plants, the pathogen causing the adlay disease was identified.The pathogen formed acervuli with 2-3 brown setae;falcate single-celled spores 20 × 3-5μ m in size;elliptic or round appressoria and brown sclerotia. It was different in morphological characters and host range from the four currently identified Colletotrichum species infecting graminaceous plant species.A 565 bp rDNA sequence of the pathogen was amplified through PCR (GenBank accession number: DQ539441). BLAST search based on this sequence suggested that this pathogen is most closely related to the graminaceous plant species-infecting pathogen C. caudatum. However, sequence difference between the two fungi was significant for ITS1 with 25 out of total 183-bp, although not for ITS2 with only 4 out of total 160-bp.Data from morphological study and ITS sequence analysis demonstrate that thepathogen causing adlay disease is a species of Colletotrichum. Adlay is a newly reported host of Colletotrichum. The species and its position in Colletotrichum classification system remain to be studied.2. Nonhost resistance of rice to C. gloeosporioides was analysed. Rice is a nonhost of C. gloeosporioides pepper isolate. No symptom was observed after inoculation with spore suspensions of this fungus. Conidiospores on the surface of rice leaves after inoculation germinated normally with germ tuber. Appressoria formed from germ tubes and normally melanized. However, no growth of internal hyphae inside the plant cells was observed. Germ tubes were very long, reaching 120-130 um, which, was nearly three times as long as those in the surface of host plant pepper. Furthermore, most appressoria formed in nonhost rice was irregularly shaped, and some of the germ tubes formed two appressoria, while all appressoria formed in host pepper was elliptic or round, and no germ tubes formed two appressoria.Time-course changes of NO level during rice - C. gloeosporioides interaction were investigated. One significant peak was observed after inoculation with both C. gloeosporioides and Magnaporthe grisea, however, the timing reaching the peak and the amount of the peak varied during different interactions. NO level during rice - C. gloeosporioides nonhost resistance interaction increased as early as 4 h post inoculation (hpi) with C. gloeosporioides, then kept increasing, reached the peak 1.15 mg/Kg fresh weight, a value 2.2 times as high as control, 36 hpi, then decreased to the level similar to water-treated control 48 hpi and maintained henceforth. NO level during rice - M. grisea host incompatible interaction attained the peak 1.08 mg/Kg fresh weight, which was 1.9 times as high as control, 60 hpi. While NO level during both rice - M. grisea and pepper- C. gloeosporioides host compatible interactions got the peak 0.95 mg/Kg fresh weight, a value 1.9 times as high as control, 96 hpi.3. The role of NO synthase (NOS) in resistance to Tobacco rattle virus (TRV) and C/"-4-dependent HR was studied employing virus-induced gene silencing (VIGS) technique. NOS1 cDNA fragments were cloned from tobacco and tomato (GenBank accession numbers: NtNOSl, DQ539435;LeNOSl, DQ539436). The NtNOSl fragment was sub-cloned into the TRV VIGS vector pYL156, and subjected to Agrobacterium-mediated VIGS analyses. It was found that M9S7-silenced plants showed obvious TRV viral symptoms, and the severity of the symptoms was relatedwith the agro-inoculation methods. Plants with severe symptoms died 3-4 weeks post agro-inoculation. No obvious viral symptom was observed in non-silenced control plants. Real Time-PCR analysis revealed that transcripts of TRV coat protein gene accumulated to a much higher level in M?S7-silenced plants than in non-silenced control plants, with the peak in the former 4.1 times as high as in controls. These data indicate that NOSl might play an important role in resistance to TRV.HR test was conducted by agro-infiltration with Agrobacterium suspensions mixed at a 1:1 ratio with those expressing Cf-4 and Avr4 respectively. No difference was observed in both time-course and severity of HR necrosis in between NOSl -silenced plants and non-silenced control plants. Thus, silencing of NOSl does not affect Cf-4/Avr4-dependent HR.