Ralstonia solanacearum causes a lethal bacterial wilt disease in many crops, leading to huge losses in crop production every year.  Understanding of plant–R. solanacearum interactions will aid to develop efficient strategies to control the disease.  As a soilborne pathogen, R. solanacearum naturally infects plants via roots.  A huge limitation in studying plant–R. solanacearum interactions is the large variation of R. solanacearum infection assay due to the variable soil conditions and uneven inoculum exposure.  Here, we developed a robust and reliable Petri-dish inoculation method which allows consistent and stable infection in young plant seedlings.  This method is easy to use, takes about only 10 days from seed germination to the completion of inoculation assay, and requires less inoculum of bacteria as well as growth chamber space.  We proved the efficacy of the seedling Petri-dish inoculation method by analyzing plant defense primed by molecular patterns, resistance of defense-related plant mutants, and virulence of R. solanacearum mutants.  Furthermore, we demonstrated that the seedling Petri-dish inoculation method can be applied to other host plants such as tobacco and has great potential for high-throughput screening of resistant plant germplasms to bacterial wilt in the future.

Meloidogyne vitis is a new root-knot nematode parasitic on grape root in Yunnan Province, China.  In order to establish a rapid, reliable and specific molecular detection method for M. vitis, the species-specific primers were designed with rDNA-ITS (ribosomal DNA internal transcribed spacer) gene fragment as the target.  The reaction system was optimized and the reliability, specificity and sensitivity of primer were testified, therefore, a rapid PCR detection method for M. vitis was established.  The result showed that the optimal annealing temperature of the primers was 53°C, which was suitable for the detection of different life stages of M. vitis.  Specificity test showed that the specific fragment size of 174 bp was obtained from M. vitis, but other five non-target nematodes did not have any amplification bands, thus effectively distinguish M. vitis and the other five species, and could specifically detect the M. vitis from mixed populations.  Sensitivity test showed that this PCR technique could detect the DNA of a single second-stage juvenile (J₂) and 10^–4 female.  Futhermore, this PCR technique could be used to detect directly M. vitis from soil samples.  The rapid, sensitive and specific PCR molecular detection technique could be used for the direct identification of a single J₂ of M. vitis and the detection of M. vitis in mixed nematode populations and the detection of two J₂s or one male in 0.5 g soil samples, which will provide technical support for the investigation of the occurrence and damage of M. vitis and the formulation of efficient green control strategies.

Cereal cyst nematode (Heterodera avenae, CCN) distributes worldwide and has caused severe damage to cereal crops, and a model host will greatly aid in the study of this nematode.  In this research, we assessed the sensitivity of 25 inbred lines of Brachypodium distachyon to H. avenae from Beijing, China.  All lines of B. distachyon were infested by second-stage juveniles (J2s) of H. avenae from Daxing District of Beijing population, but only 13 inbred lines reproduced 0.2–3 cysts/plant, showing resistance.  The entire root system of the infested B. distachyon appeared smaller and the fibrous roots were shorter and less numerous.  We found that a dose of 1 000 J2s of H. avenae was sufficient for nematode infestation.  We showed that Koz-1 of B. distachyon could reproduce more cysts than TR2A line.  Line Koz-1 also supported the complete life cycles of 5 CCN geographical populations belonging to the Ha1 or Ha3 pathotype group.  Our results suggest that B. distachyon is a host for CCN.

Streptomyces rubrogriseus HDZ-9-47, isolated from eggs of Meloidogyne spp., was evaluated as a potential biocontrol agent of Meloidogyne incognita under in vitro and protective field.  Microscopic observations showed that HDZ-9-47 parasitized eggs of M. incognita within 7 days.  In vitro, the culture filtrate of HDZ-9-47 caused 97.0% mortality of second-stage juveniles (J2s) of M. incognita and inhibited more than 50% egg hatching.  In the field, compared with the control, the root-knot index and J2s density in the treatment of drench the broth contained 10¹² HDZ-9-47 spores were respectively reduced by 51.1 and 80.7% at 90 days post transplantation, which were better than that in other application doses and methods.  In addition, reduction rates of root-knot index and J2s density of the treatment of combined application of HDZ-9-47 with biofumigation was 87.1 and 91.0%, respectively, better than either of HDZ-9-47 or biofumigation used alone or fosthiazate treatment.  And tomato yield also increased by 16.1%.  Together, our results suggest that HDZ-9-47 could be an effective biocontrol agent of M. incognita, and that application of HDZ-9-47 combined with cabbage residue biofumigation was a promising and sustainable option for M. incognita control.

Pasteurella multocida, a Gram-negative nonmotile coccobacillus, is the causative agent of fowl cholera, bovine hemorrhagic septicemia, enzoonotic pneumonia and swine atropic rhinitis. Two filamentous hemagglutinin genes, fhaB1 and fhaB2, are the potential virulence factors. In this study, an inactivation fhaB1 mutant of P. multocida in avian strain C48-102 was constructed by a kanamycin-resistance cassette. The virulence of the fhaB1 mutant and the wild type strain was assessed in chickens by intranasal and intramuscular challenge. The inactivation of fhaB1 resulted in a high degree of attenuation when the chickens were challenged intranasally and a lesser degree when challenged intramuscularly. The fhaB1 mutant and the wild type strain were investigated their sensitivity to the antibody-dependent classical complement-mediated killing pathway in 90% convalescent chicken serum. The fhaB1 mutant was serum sensitive as the viability has reduced between untreated serum and heat inactivated chicken serum (P<0.007). These results confirmed that FhaB1 played the critical roles in the bacterial pathogenesis and further studies were needed to investigate the mechanism which caused reduced virulence of the fhaB1 mutant.